Modified factor ix, and compositions, methods and uses for gene transfer to cells, organs, and tissues

ABSTRACT

The invention relates to modified Factor IX coding sequence, expression cassette, vectors such as viral (e.g., lenti- or adeno-associated viral) vectors, and gene transfer methods and uses. In particular, to target Factor IX nucleic acid to cells, tissues or organs for expression (transcription) of Factor IX.

RELATED APPLICATIONS

This patent application is a continuation application of U.S. patentapplication no. 17,014,782, filed Sep. 8, 2020, which is a continuationapplication of U.S. patent application Ser. No. 15/191,357, filed Jun.23, 2016, now U.S. Pat. No. 10,799,566, issued Oct. 13, 2020, whichclaims the benefit of U.S. patent application No. 62/183,599, filed Jun.23, 2015, application No. 62/315,453, filed Mar. 30, 2016, applicationNo. 62/338,315, filed May 18, 2016, application No. 62/348,781, filedJun. 10, 2016 and application No. 62/349,572, filed Jun. 13, 2016, allof which applications are expressly incorporated herein by reference intheir entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jul. 9, 2021, is named“CHOP0561110_ST25.txt” and is 108,457 bytes in size.

INTRODUCTION

Genetic disorders, caused by absence or a defect in a desirable gene(loss of function) or expression of an undesirable or defective gene(gain of function) lead to a variety of diseases. One example of a lossof function genetic disorder is hemophilia, an inherited bleedingdisorder caused by deficiency in either coagulation factor VIII (FVIII,hemophilia A) or factor IX (FIX, hemophilia B). One example of a gain offunction genetic disorder is Huntington's disease, a disease caused by apathologic “HTT” gene (encodes the huntingtin protein) that encodes amutated protein that accumulates within and leads to gradual destructionof neurons, particularly in the basal ganglia and the cerebral cortex.

Current treatment for hemophilia consists in the intravenousadministration of recombinant clotting factor either on demand, in casea bleeding occurs, or prophylactically. However, this therapeuticapproach has several drawbacks such as the need for repeated infusions,the cost of the treatment, the risk of developing anti-therapeuticfactor immune responses, and the risk of potentially fatal bleedings.These limitations have prompted the development of gene-based therapiesfor hemophilia. To this end, hemophilia is ideal for gene transfer basedtherapy as 1) the therapeutic window is very wide, as levels just above1% of normal already can result in a change in phenotype from severe tomoderate, and levels of 100% are not associated to any side effects; 2)tissue specific expression of the therapeutic transgene is not strictlyrequired; and 3) there is a considerable experience in measuring theendpoints of therapeutic efficacy.

Currently, adeno-associated virus (AAV) vectors are recognized as thegene transfer vectors of choice since they have the best safety andefficacy profile for the delivery of genes in vivo. Of the AAV serotypesisolated so far, AAV2 and AAV8 have been used to target the liver ofhumans affected by severe hemophilia B.

SUMMARY

The invention provides nucleic acid sequences, expression vectors (e.g.,vector genomes) and plasmids, compositions and viral vectors in whichthe nucleic acid encodes Factor IX (e.g., human Factor IX). The nucleicacid encoding Factor IX is modified to reduce the number of CpG(cytosine-guanine) dinucleotides relative to a comparison FIX codingsequence. In a particular embodiment, a modified nucleic acid encodingFactor IX has a reduced number of CpG di-nucleotides compared to awild-type or native sequence encoding human Factor IX.

Modified nucleic acids encoding Factor IX, such as FIX modified toreduce the number of CpG (cytosine-guanine) dinucleotides, can beincluded in vectors, such as viral vectors. Representative viral vectorsinclude lenti- and parvo-viral vectors (e.g., adenoviral oradeno-associated virus (AAV) vectors) which target, for example,hepatocyte cells of the liver, among other cell types. As a vector fornucleic acid sequence delivery, AAV vectors drive expression of thepolynucleotide in cells. Polynucleotides that encode proteins, such as amodified nucleic acid encoding Factor IX, are able to be expressed afteradministration, optionally at therapeutic levels.

Accordingly, there are provided recombinant AAV vectors that include(encapsidate, package) vector genomes that include a modified nucleicacid encoding Factor IX. In particular embodiments, a recombinant AAVparticle encapsidates or packages a vector genome. Such inventionrecombinant AAV particles include a viral vector genome which includes aheterologous polynucleotide sequence (e.g., modified nucleic acidencoding Factor IX, such as FIX modified to reduce the number of CpG(cytosine-guanine) dinucleotides). In one embodiment, a vector genomethat includes a modified nucleic acid encoding Factor IX, such as FIXmodified to reduce the number of CpG (cytosine-guanine) dinucleotides isencapsidated or packaged by an AAV capsid or an AAV capsid variant.

In the invention recombinant AAV vectors, the heterologouspolynucleotide sequence may be transcribed and subsequently translatedinto a protein. In various aspects, the heterologous polynucleotidesequence encodes a therapeutic protein. In particular aspects, theprotein is a blood clotting factor (e.g., Factor IX, Factor XIII, FactorX, Factor VIII, Factor VIIa, or protein C). In more particular aspects,the vector includes a modified nucleic acid encoding Factor IX (e.g.,modified nucleic acid encoding Factor IX, such as FIX modified to reducethe number of CpG (cytosine-guanine) dinucleotides).

AAV and AAV variants such as capsid variants can deliver polynucleotidesand/or proteins that provide a desirable or therapeutic benefit, therebytreating a variety of diseases. For example, AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, andvariants thereof and AAV capsid variants (e.g., 4-1) are useful vectorsto deliver to cells, tissues and organs, therapeutic genes (e.g., FactorIX) to treat hemophilia A, B, etc.

In the invention recombinant viral and AAV vectors that include(encapsidate, package) vector genome (viral or AAV) include additionalelements that function in cis or in trans. In particular embodiments, arecombinant viral (e.g., AAV) vector that includes (encapsidate,package) a vector genome also has: one or more inverted terminal repeat(ITR) sequences that flank the 5′ or 3′ terminus of the heterologouspolynucleotide sequence (e.g., modified nucleic acid encoding Factor IX,such as FIX modified to reduce the number of CpG (cytosine-guanine)dinucleotides); an expression control element that drives transcription(e.g., a promoter or enhancer) of the heterologous polynucleotidesequence (e.g., modified nucleic acid encoding Factor IX, such as FIXmodified to reduce the number of CpG (cytosine-guanine) dinucleotides),such as a constitutive or regulatable control element, ortissue-specific expression control element; an intron sequence, astuffer or filler polynucleotide sequence; and/or a poly-adenylationsequence located 3′ of the heterologous polynucleotide sequence.

Accordingly, vectors can further include an intron, an expressioncontrol element (e.g., a constitutive or regulatable control element, ora tissue-specific expression control element or promoter such as forliver expression, e.g., a human α₁-anti-trypsin (hAAT) Promoter and/orapolipoprotein E (ApoE) HCR-1 and/or HCR-2 enhancer), one or moreadeno-associated virus (AAV) inverted terminal repeats (ITRs) (e.g., anITR sequence of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes) and/or a fillerpolynucleotide sequence. Position of such additional elements can vary.In particular aspects, an intron is within the sequence encoding humanFIX protein, and/or the expression control element is operably linked tothe sequence encoding human FIX protein, and/or the AAV ITR(s) flanksthe 5′ or 3′end of the sequence encoding human FIX protein, and/orwherein the filler polynucleotide sequence flanks the 5′ or 3′end of thesequence encoding human FIX protein.

In various embodiments, a FIX nucleic acid modified to reduce the numberof CpG (cytosine-guanine) dinucleotides can have 1-5 fewer, 5-10 fewer,10-15 fewer, 15-20 fewer, 20-25 fewer, 25-30 fewer, 30-40 fewer, 40-55,55-75, fewer, 75-100, 100-150 fewer, 150-200 fewer CpG di-nucleotidesthan native or wild-type sequence encoding human Factor IX. Inparticular aspects, a FIX nucleic acid modified as set forth hereinencodes human FIX protein which is expressed at levels greater than orcomparable to a wild-type or native sequence encoding human Factor IXnot having a reduced number of CpG di-nucleotides.

In additional embodiments, an intron, an expression control element(e.g., a constitutive or regulatable control element, or atissue-specific expression control element or promoter such as for liverexpression, e.g., a human α₁-anti-trypsin (hAAT) Promoter and/orapolipoprotein E (ApoE) HCR-1 and/or HCR-2 enhancer), one or moreadeno-associated virus (AAV) inverted terminal repeats (ITRs) (e.g., anITR sequence of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes) and/or a fillerpolynucleotide sequence can be modified to reduce the number of CpG(cytosine-guanine) dinucleotides as compared to a counterpart native orwild-type expression control element, adeno-associated virus (AAV)inverted terminal repeats (ITRs) and/or a filler polynucleotidesequence. In particular aspects, an expression control element,adeno-associated virus (AAV) inverted terminal repeat (ITR) and/or afiller polynucleotide sequence has 1-5 fewer, 5-10 fewer, 10-15 fewer,15-20 fewer, 20-25 fewer, 25-30 fewer, 30-40 fewer, 40-55, 55-75, fewer,75-100, 100-150 fewer, 150-200 fewer CpG di-nucleotides than native orwild-type counterpart sequence.

Exemplary AAV vectors include AAV capsid sequence of any of AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 orAAV-2i8, or a capsid variant of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8. Recombinant AAVparticles of the invention also include AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, andvariants thereof. Particular capsid variants include capsid variants ofsuch as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, Rh10, Rh74 or AAV-2i8, such as a capsid sequence with an aminoacid substitution, deletion or insertion/addition. In particularaspects, a substitution is made in the Rh74 VP1 capsid sequence (SEQ IDNO:1), for example at any one of amino acid positions 195, 199, 201 or202. In more particular aspects, substituted residues correspond to anA, V, P or N amino acid at any one of amino acid positions 195, 199, 201or 202 of Rh74 VP1 capsid sequence. In further particular aspects, thecapsid sequence has an A residue at amino acid position 195; a V residueat amino acid positions 199, a P residue at amino acid position 201, oran N residue at amino acid position 202 of Rh74 VP1 capsid sequence. Inadditional particular aspects, the capsid sequence has any two, three orall four of the following: an A residue at amino acid position 195; a Vresidue at amino acid positions 199, a P residue at amino acid position201, or an N residue at amino acid position 202 of Rh74 VP1 capsidsequence.

In more particular aspects, a capsid variant comprises any of SEQ IDNOS:4-9. additional embodiments, an AAV vector has a capsid sequencewith an AAV VP1, VP2 and/or VP3 sequence having 90% or more sequenceidentity to any VP1, VP2 and/or VP3 of any AAV serotype. In particularaspects, an AAV vector has a capsid sequence with a VP1, VP2 and/or VP3capsid sequence having 90% or more identity to AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 VP1,VP2 and/or VP3 sequences. In more particular aspects, an AAV vector hasa capsid sequence with a VP1, VP2 or VP3 capsid sequence selected fromany of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes.

In additional embodiments, a recombinant vector genome includes amodified nucleic acid encoding Factor IX, such as FIX modified to reducethe number of CpG (cytosine-guanine) dinucleotides and a filler orstuffer polynucleotide sequence. In particular aspects, modified nucleicacid encoding Factor IX has a length less than about 4.7 kb. In furtherparticular aspects, modified nucleic acid encoding Factor IX has alength less than 4.7 kb and is flanked by one or more AAV ITRs, orpositioned within two adeno-associated virus (AAV) ITR sequences. Inadditional particular aspects, a filler or stuffer polynucleotidesequence has a length that when combined with modified nucleic acidencoding Factor IX the total combined length of the Factor IX encodingnucleic acid sequence and filler or stuffer polynucleotide sequence isbetween about 3.0-5.5 kb, or between about 4.0-5.0 kb, or between about4.3 kb-4.8 kb.

Filler or stuffer polynucleotide sequences can be located in the vectorsequence at any desired position such that it does not prevent afunction or activity of the vector. In one aspect, a filler or stufferpolynucleotide sequence is positioned between a 5′ and/or 3′ ITR (e.g.,an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, Rh10, Rh74 or AAV-2i8, and variants thereof) that flanks therespective 5′ and/or 3′ termini of a Factor IX encoding nucleic acidsequence, such as FIX with a reduced number of CpG (cytosine-guanine)dinucleotides. In another aspect, a filler or stuffer polynucleotidesequence is positioned within a 5′ and/or 3′ ITR that flanks therespective 5′ and/or 3′ termini of a Factor IX encoding nucleic acidsequence, such as FIX with a reduced number of CpG dinucleotides. In anadditional aspect, a filler or stuffer polynucleotide sequence ispositioned adjacent to 5′ and/or 3′ ITR that flanks the respective 5′and/or 3′ termini of a Factor IX encoding nucleic acid sequence, such asFIX with a reduced number of CpG dinucleotides. In a further aspect, afiller or stuffer polynucleotide sequence is positioned within amodified nucleic acid encoding Factor IX, such as FIX with a reducednumber of CpG dinucleotides, e.g., analogous to an intron within agenomic nucleic acid.

Accordingly, in various embodiments, a filler or stuffer polynucleotidesequence is positioned adjacent to an AAV ITR sequence; positionedwithin two adeno-associated virus (AAV) ITR sequences; positionedoutside two adeno-associated virus (AAV) ITR sequences; or there are twofiller or stuffer polynucleotide sequences, a first filler or stufferpolynucleotide sequence positioned within two adeno-associated virus(AAV) ITR sequences, and a second filler or stuffer polynucleotidesequence positioned outside two adeno-associated virus (AAV) ITRsequences.

In more particular aspects, a filler or stuffer polynucleotide sequencehas a length that when combined with a modified nucleic acid encodingFactor IX, such as FIX with a reduced number of CpG dinucleotides, thetotal combined length of the heterologous polynucleotide sequence andfiller or stuffer polynucleotide sequence is between about 3.0 kb-5.5kb, between about 4.0-5.0 kb, or between about 4.3 kb-4.8 kb, whenpositioned within two adeno-associated virus (AAV) ITR sequences. Inother more particular aspects, a filler or stuffer polynucleotidesequence has a length greater than 4.7 kb, between about 5.0-10.0 kb, orbetween about 6.0-8.0 kb, when positioned outside two adeno-associatedvirus (AAV) ITR sequences.

Typically, a filler or stuffer polynucleotide sequence is inert orinnocuous and has no function or activity. In various particularaspects, a filler or stuffer polynucleotide sequence is not a bacterialpolynucleotide sequence, a filler or stuffer polynucleotide sequence isnot a sequence that encodes a protein or peptide, a filler or stufferpolynucleotide sequence is a sequence distinct from any of: theheterologous polynucleotide sequence (e.g., modified nucleic acidencoding Factor IX), an AAV inverted terminal repeat (ITR) sequence, anexpression control element, an origin of replication, a selectablemarker or a poly-adenylation (poly-A) signal sequence.

In various additional particular aspects, a filler or stufferpolynucleotide sequence is an intron sequence that is related to orunrelated to the heterologous polynucleotide sequence (e.g., modifiednucleic acid encoding Factor IX, such as FIX with a reduced number ofCpG dinucleotides). In particular aspects, the intron sequence ispositioned within the heterologous polynucleotide sequence (e.g.,modified nucleic acid encoding Factor IX, such as FIX with a reducednumber of CpG dinucleotides). In other particular aspects, the intronsequence is related to the heterologous polynucleotide sequence (e.g.,modified nucleic acid encoding Factor IX, such as FIX with a reducednumber of CpG dinucleotides) as the intron is in genomic DNA, such asthe genomic DNA that encodes a protein which protein is also encoded bythe heterologous polynucleotide sequence (e.g., modified nucleic acidencoding Factor IX).

Invention recombinant lenti- and parvo-virus (e.g., AAV) vectors such asAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,Rh10, Rh74 and AAV-2i8, and variants such as capsid variant (e.g., 4-1)particles that include (encapsidate, package) recombinant AAV vectorgenome can be included within cells. In such embodiments, cells cancomprise packaging cells that produce or can be lysed to produce virus(AAV) particles, or target cells in which it is desired to express theheterologous polynucleotide sequence. Accordingly, cells that comprisemodified Factor IX encoding nucleic acid, such as FIX with a reducednumber of CpG dinucleotides, vectors and lenti- and parvo-virus (e.g.,AAV) vectors such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8, and variants (e.g., such as4-1) particles are provided.

In additional embodiments, a nucleic acid sequence encoding human FIXprotein, wherein said nucleic acid has a reduced number of CpGdi-nucleotides compared to native sequence encoding human Factor IX, oran expression vector or plasmid comprising a nucleic acid sequenceencoding human FIX protein, wherein said nucleic acid has a reducednumber of CpG di-nucleotides compared to native sequence encoding humanFactor IX is include in a composition. In a particular aspect, suchnucleic acid sequences can be included in a pharmaceutical composition.Accordingly, invention recombinant lenti- and parvo-virus (e.g., AAV)vectors such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, Rh10, Rh74 and AAV-2i8, and variants (e.g., such as 4-1)particles that include (encapsidate, package) vector genome can beincluded within pharmaceutical compositions. Such compositions areuseful for administration of recombinant vector (e.g., AAV) and virusparticles such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, Rh10, Rh74 or AAV-2i8, and variants (e.g., 4-1) thatinclude (encapsidate, package) vector (e.g., AAV) genomes to a subject.

Invention recombinant lenti- and parvo-virus (e.g., AAV) vectors such asAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,Rh10, Rh74 and AAV-2i8, and variant (e.g., such as 4-1) particles thatinclude (encapsidate, package) vector genome may be employed in variousmethods and uses. Accordingly, there are provided methods and uses fordelivering or transferring a heterologous polynucleotide sequence (e.g.,modified nucleic acid encoding Factor IX, such as FIX with a reducednumber of CpG dinucleotides) into an organism or cell, such as a mammalor a cell of a mammal.

In one embodiment, a method or use includes administering a lenti- orparvo-virus (e.g., AAV) vector such as AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8, or variantthat includes a heterologous polynucleotide sequence (e.g., modifiednucleic acid encoding Factor IX such as FIX with a reduced number of CpGdinucleotides) particle in a vector genome (encapsidate, package) to amammal or a cell of a mammal under suitable conditions to deliver ortransfer the heterologous polynucleotide sequence into the mammal or thecell of a mammal. In one aspect, the method or use transfers/deliversthe heterologous polynucleotide (e.g., modified nucleic acid encodingFactor IX, such as FIX with a reduced number of CpG dinucleotides) intothe mammal and/or cell. In another aspect, the method allowstransfer/delivery of the heterologous polynucleotide (e.g., modifiednucleic acid encoding Factor IX, such as FIX with a reduced number ofCpG dinucleotides) into the cell, subsequent transcription to form atranscript and subsequent translation to form a gene product (e.g.,Factor IX).

In additional embodiments, a method or use is for treating a subject(e.g., mammal) deficient or in need of protein expression or functionincludes providing a lenti- or parvo-virus (e.g., AAV) vector such asAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,Rh10, Rh74 or AAV-2i8, or a variant, a plurality of such viral (e.g.,AAV) particles, or a pharmaceutical composition of lenti- or parvo-virus(e.g., AAV) vectors such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, or variant, particle orplurality of such viral (e.g., AAV) particles; and administering theviral particle, plurality of viral particles, or pharmaceuticalcomposition of viral particles or plurality of viral particles to thesubject (e.g., mammal). The heterologous polynucleotide sequence (e.g.,modified nucleic acid encoding Factor IX, such as FIX with a reducednumber of CpG dinucleotides) so administered may be subsequentlyexpressed in the subject (e.g., mammal).

Methods and uses for administration or delivery include any modecompatible with a subject. In particular embodiments, a lenti- orparvo-virus (e.g., AAV) vector such as AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8, andvariants, or plurality of such viral particles is administered ordelivered parenterally, such as intravenously, intraarterially,intramuscularly, subcutaneously, or via catheter.

Subjects include mammals, such as humans and non-humans (e.g.,primates). In particular embodiments, a subject would benefit from or isin need of expression of a heterologous polynucleotide sequence. In amore particular embodiment, a subject would benefit from Factor IXexpression or function, e.g., such as a subject that expresses reducedamount of Factor IX, such as a subject with hemophilia B.

In accordance with the invention, methods of producing recombinantlenti- and parvo-virus (e.g., AAV) vectors such as AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 andAAV-2i8, and variants, that include (encapsidate, package) vector genomeare provided. In one embodiment, a method includes introducing into apackaging cell a recombinant vector (e.g., AAV) plasmid to produce aproductive viral infection; and culturing the packaging cells underconditions to produce recombinant viral particles. In anotherembodiment, a method of producing recombinant viral or AAV particleswith reduced amounts of recombinant viral particles in which therecombinant viral vector includes contaminating nucleic acid, includesintroducing into a packaging cell a recombinant vector (e.g., AAV)plasmid; and culturing the packaging cells under conditions to producerecombinant viral particles, wherein the recombinant viral particlesproduced have reduced numbers of viral particles with vector genomesthat contain contaminating nucleic acid compared to the numbers of viralparticles that contain contaminating nucleic acid produced underconditions in which a filler or stuffer polynucleotide sequence isabsent from the recombinant viral vector. In particular aspects, thecontaminating nucleic acid is bacterial nucleic acid; or a sequencesother than the heterologous polynucleotide sequence, or ITR, promoter,enhancer, origin of replication, poly-A sequence, or selectable marker.

Packaging cells include mammalian cells. In particular embodiments, apackaging cell includes helper (e.g., AAV) functions to package the(heterologous polynucleotide) sequence (e.g., modified nucleic acidencoding Factor IX, such as FIX with a reduced number of CpGdinucleotides), expression vector (e.g., vector genome), into a viralparticle (e.g., AAV particle). In particular aspects, a packaging cellprovides AAV Rep and/or Cap proteins (e.g., Rep78 or/and Rep68proteins); a packaging cell is stably or transiently transfected withpolynucleotide(s) encoding Rep and/or Cap protein sequence(s); and/or apackaging cell is stably or transiently transfected with Rep78 and/orRep68 protein polynucleotide encoding sequence(s).

In the invention recombinant lenti- or parvo-virus (e.g., AAV) vectors,and accompanying cis (e.g., expression control elements, ITRs, polyA,)or trans (e.g., capsid proteins, packaging functions such as Rep/Capprotein) elements can be based upon any organism, species, strain orserotype. Invention recombinant viral (e.g., AAV) particles aretypically based upon AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8, and variants, but alsoinclude hybrids or chimeras of different serotypes. Representative AAVserotypes include, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8 serotypes.Accordingly, invention recombinant viral (e.g., AAV) particlescomprising vector genomes can include a capsid protein from a differentserotype, a mixture of serotypes, or hybrids or chimeras of differentserotypes, such as a VP1, VP2 or VP3 capsid protein of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8serotype. Furthermore, invention recombinant lenti- or parvo-virus(e.g., AAV) vectors such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8, sequences, plasmids,vector genomes, can include elements from any one serotype, a mixture ofserotypes, or hybrids or chimeras of different serotypes. In variousembodiments, a recombinant AAV vector includes a, ITR, Cap, Rep, and/orsequence derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 and/or AAV-2i8 serotype, or a mixture,hybrid or chimera of any of the foregoing AAV serotypes.

ADDITIONAL EMBODIMENTS OF THE DISCLOSURE

Embodiment 1. A nucleic acid sequence encoding human Factor IX protein,wherein said nucleic acid has a reduced number of CpG di-nucleotidescompared to a wild-type sequence encoding human Factor IX.

Embodiment 2. An expression vector or plasmid comprising a nucleic acidsequence encoding human Factor IX protein, wherein said nucleic acid hasa reduced number of CpG di-nucleotides compared to native sequenceencoding human Factor IX, and/or wherein if one or more sequencesadditional to the nucleic acid sequence encoding human Factor IX arepresent in said vector or plasmid said additional sequences optionallyhave a reduced number of CpG di-nucleotides compared to a counterpartnative or wild-type sequence.

Embodiment 3. The nucleic acid sequence encoding human Factor IX proteinof Embodiment 1, expression vector or plasmid of Embodiment 2, orcomposition of Embodiment 3, further comprising an intron, an expressioncontrol element, one or more adeno-associated virus (AAV) invertedterminal repeats (ITRs) and/or a filler polynucleotide sequence,optionally wherein said intron, expression control element,adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/orfiller polynucleotide sequence has a reduced number of CpGdi-nucleotides compared to a counterpart native or wild-type expressioncontrol element, adeno-associated virus (AAV) inverted terminal repeats(ITRs) and/or filler polynucleotide sequence.

Embodiment 4. The nucleic acid sequence encoding human Factor IX proteinof Embodiment 4, wherein the intron is within the sequence encodinghuman Factor IX protein, or wherein the expression control element isoperably linked to the sequence encoding human Factor IX protein, orwherein the AAV ITR(s) flanks the 5′ or 3′end of the sequence encodinghuman Factor IX protein, or wherein the filler polynucleotide sequenceflanks the 5′ or 3′end of the sequence encoding human Factor IX protein.

Embodiment 5. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 1-5 fewer CpG di-nucleotides than native sequence encodinghuman Factor IX.

Embodiment 6. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 5-10 fewer CpG di-nucleotides than native sequence encodinghuman Factor IX.

Embodiment 7. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 10-15 fewer CpG di-nucleotides than native sequenceencoding human Factor IX.

Embodiment 8. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 15-20 fewer CpG di-nucleotides than native sequenceencoding human Factor IX.

Embodiment 9. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 20-25 fewer CpG di-nucleotides than native sequenceencoding human Factor IX.

Embodiment 10. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 25-30 fewer CpG di-nucleotides than native sequenceencoding human Factor IX.

Embodiment 11. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 30-40 fewer CpG di-nucleotides than native sequenceencoding human Factor IX.

Embodiment 12. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence has 40-55 fewer CpG di-nucleotides than native sequenceencoding human Factor IX.

Embodiment 13. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX protein,intron, expression control element, ITR(s) and/or filler polynucleotidesequence is devoid of any CpG di-nucleotides.

Embodiment 14. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX proteincomprises a sequence with 80% or more identity to SEQ ID NO:10, andencodes functional Factor IX as determined by a clotting assay.

Embodiment 15. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX proteincomprises a sequence with 90% or more identity to SEQ ID NO:10, andencodes functional Factor IX as determined by a clotting assay.

Embodiment 16. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX proteincomprises a sequence with 95% or more identity to SEQ ID NO:10, andencodes functional Factor IX as determined by a clotting assay.

Embodiment 17. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX proteincomprises SEQ ID NOs:10, 25 or 26.

Embodiment 18. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the native sequence encoding humanFactor IX comprises the sequence set forth in SEQ ID NO:11.

Embodiment 19. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the intron sequence comprises thesequence set forth in SEQ ID NO:17.

Embodiment 20. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the expression control element comprisesan enhancer sequence comprising the sequence set forth as SEQ ID NO:14.

Embodiment 21. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the expression control element comprisesa promoter sequence comprising the sequence set forth as SEQ ID NO:15.

Embodiment 22. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence of the one or moreadeno-associated virus (AAV) inverted terminal repeats (ITRs) comprisesthe sequence set forth as SEQ ID NO:13 and/or SEQ ID NO:20.

Embodiment 23. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the filler polynucleotide sequencecomprises a sequence set forth as SEQ ID NO:21.

Embodiment 24. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX proteinhas reduced ability to induce an immune response.

Embodiment 25. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the sequence encoding human FIX proteinis expressed at levels greater than or comparable to native sequenceencoding human Factor IX not having a reduced number of CpGdi-nucleotides.

Embodiment 26. The nucleic acid sequence encoding human Factor IXprotein of any of Embodiments 1-5, wherein the expression controlelement comprises a constitutive or regulatable control element, or atissue-specific expression control element or promoter.

Embodiment 27. The nucleic acid sequence encoding human Factor IXprotein of any of Embodiments 1-5, wherein the expression controlelement comprises a an element that confers expression in liver.

Embodiment 28. The nucleic acid sequence encoding human FIX protein ofany of Embodiments 1-5, wherein the expression control element comprisesa human al-anti-trypsin (hAAT) Promoter and/or apolipoprotein E (ApoE)HCR-1 and/or HCR-2 enhancer.

Embodiment 29. The nucleic acid sequence encoding human Factor IXprotein of any of Embodiments 1-5, further comprising a poly-adenylationsequence located 3′ of the nucleic acid sequence encoding human FactorIX.

Embodiment 30. The nucleic acid sequence encoding human Factor IXprotein of Embodiment 29, wherein the poly-adenylation sequence islocated 3′ of the nucleic acid sequence encoding human Factor IXcomprises a bGH poly-adenylation sequence.

Embodiment 31. The nucleic acid sequence encoding human FIX protein ofEmbodiment 29, wherein the poly adenylation sequence located 3′ of thenucleic acid sequence encoding human Factor IX comprises apoly-adenylation sequence having all CpG di-nucleotides removedtherefrom.

Embodiment 32. The nucleic acid sequence encoding human FIX protein ofEmbodiment 31, wherein the poly-adenylation sequence comprises thesequence set forth as SEQ ID NO:19.

Embodiment 33. The nucleic acid sequence encoding human FIX protein ofEmbodiment 4 or 5, wherein the filler polynucleotide sequence is located3′ of the sequence encoding human FIX protein.

Embodiment 34. The nucleic acid sequence encoding human FIX protein ofEmbodiment 4 or 5, wherein the AAV ITR(s) flanks the 3′end of thesequence encoding human FIX protein.

Embodiment 35. The nucleic acid sequence encoding human FIX protein ofEmbodiment 34, wherein the filler polynucleotide sequence is located 3′of the AAV ITR(s) flanking the 3′end of the sequence encoding human FIXprotein.

Embodiment 36. The nucleic acid sequence encoding human FIX protein ofEmbodiment 4 or 5, wherein the filler polynucleotide sequence comprisesa lambda phage sequence.

Embodiment 37. A plasmid sequence encoding human FIX protein comprisingthe nucleic acid sequence encoding human FIX protein of any ofEmbodiments 1-36, further comprising one or more origins of replicationand/or a nucleic acid encoding resistance to an antibiotic.

Embodiment 38. A plasmid sequence encoding human FIX protein comprisingSEQ ID NO:12 or 26.

Embodiment 39. A viral vector comprising the sequence encoding human FIXprotein or expression vector comprising the nucleic acid sequenceencoding human FIX protein of any of Embodiments 1-5.

Embodiment 40. A viral vector according to Embodiment 39, wherein saidviral vector is a lenti- or adeno-viral vector.

Embodiment 41. A viral vector according to Embodiment 39, wherein saidviral vector is an adeno-associated viral (AAV) vector.

Embodiment 42. The AAV vector of Embodiment 41, comprising an ITRsequence of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes.

Embodiment 43. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector comprises a VP1, VP2 and/or VP3 capsid sequencehaving 90% or more identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 VP1, VP2 and/or VP3sequences.

Embodiment 44. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector comprises a VP1, VP2 or VP3 capsid sequenceselected from any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes.

Embodiment 45. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector comprises a VP1 sequence having 90% or moresequence identity to SEQ ID NO:1, or 1-50 amino acid substitutions,deletions or additions thereto; a VP2 sequence having 90% or moresequence identity to SEQ ID NO:2, or 1-50 amino acid substitutions,deletions or additions thereto; and/or

a VP3 sequence having 90% or more sequence identity to SEQ ID NO:3, or1-50 amino acid substitutions, deletions or additions thereto.

Embodiment 46. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector has an amino acid substitution at any one ofamino acid positions 195, 199, 201 or 202, of the VP1 capsid sequenceset forth as SEQ ID NO:1, or an amino acid substitution of an argininefor a lysine in the VP1 capsid sequence set forth as SEQ ID NO:1.

Embodiment 47. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector has residues of any of A, V, P or N amino acidsat any one of amino acid positions 195, 199, 201 or 202 of the VP1capsid sequence set forth as SEQ ID NO:1.

Embodiment 48. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector has an A residue at amino acid position 195; a Vresidue at amino acid positions 199, a P residue at amino acid position201, or an N residue at amino acid position 202 of the VP1 capsidsequence set forth as SEQ ID NO:1.

Embodiment 49. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector has any two, three or four of an A residue atamino acid position 195; a V residue at amino acid positions 199, a Presidue at amino acid position 201, or an N residue at amino acidposition 202 of the VP1 capsid sequence set forth as SEQ ID NO:4.

Embodiment 50. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector comprises a VP1 capsid sequence having 90% ormore identity to any of SEQ ID NOs:4-9.

Embodiment 51. The AAV vector of Embodiment 41, wherein the capsidsequence of the vector comprises a VP1 capsid sequence comprises any ofSEQ ID NOs:4-9.

Embodiment 52. A pharmaceutical composition comprising the nucleic acidsequence encoding human FIX protein of any of Embodiments 1-38 and/or aviral vector of Embodiments 39-51.

Embodiment 53. A pharmaceutical composition according to Embodiment 52,further comprising empty capsid AAV.

Embodiment 54. A pharmaceutical composition according to Embodiment 53wherein said empty capsid is selected from serotype AAV AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11.

Embodiment 55. An expression vector comprising SEQ ID NO:11 or SEQ IDNO:25.

Embodiment 56. The expression vector of Embodiment 55, furthercomprising an enhancer sequence comprising the sequence set forth as SEQID NO:14.

Embodiment 57. The expression vector of Embodiment 55, furthercomprising a promoter sequence comprising the sequence set forth as SEQID NO:15.

Embodiment 58. The expression vector of Embodiment 55, furthercomprising an upstream and/or downstream AAV2 ITR as set forth in SEQ IDNO:12 or 26, wherein said upstream ITR is positioned 5′ of the enhancerand/or said downstream AAV2 ITR is positioned 3′ of hFIX exons 2-8 setforth in SEQ ID NO:12 or 26.

Embodiment 59. The expression vector of Embodiment 55, furthercomprising a polyA sequence positioned 3′ of the hFIX exons 2-8 as setforth in SEQ ID NO:12 or 26 and positioned 5′ of the downstream AAV2ITR.

Embodiment 60. An AAV vector comprising the expression vector of any ofEmbodiments 55-59.

Embodiment 61. The AAV vector of Embodiment 60, wherein the capsidsequence comprises a VP1 capsid sequence comprises any of SEQ IDNOs:4-9.

Embodiment 62. A method for delivering or transferring a nucleic acidsequence into a cell, comprising contacting the nucleic acid sequence,expression vector, or virus vector of any of Embodiments 1-61 to saidmammalian cell, under conditions allowing transduction of said cell,thereby delivering or transferring the nucleic acid sequence into themammalian cell.

Embodiment 63. A method for delivering or transferring a nucleic acidsequence into a mammal or a cell of a mammal, comprising administeringthe nucleic acid sequence, expression vector, or virus vector of any ofEmbodiments 1-61 to said mammal or a cell of said mammal, therebydelivering or transferring the nucleic acid sequence into the mammal orcell of the mammal.

Embodiment 64. A method of treating a mammal in need of Factor IXprotein, comprising: (a) providing a nucleic acid sequence, expressionvector, or virus vector of any of Embodiments 1-61; and (b)administering an amount of the nucleic acid sequence, expression vector,or virus vector of any of Embodiments 1-61 to the mammal wherein saidFactor IX is expressed in the mammal.

Embodiment 65. The method of any of Embodiments 62-64, wherein saidFactor XI protein is expressed in a cell, tissue or organ of saidmammal.

Embodiment 66. The method of Embodiment 65, wherein the cell comprises asecretory cell.

Embodiment 67. The method of Embodiment 65, wherein the cell comprisesan endocrine cell.

Embodiment 68. The method of Embodiment 65, wherein the cell compriseshepatocyte, a neural cell, a glial cell, a retinal cell, an epithelialcell, a lung cell or a totipotent, pluripotent or multipotent stem cell.

Embodiment 69. The method of Embodiment 65, wherein the tissue or organof said mammal comprises liver, brain, central nervous system, spinalcord, eye, retina or lung.

Embodiment 70. The method of any of Embodiments 62-69, wherein themammal produces an insufficient amount of Factor IX protein, or adefective or aberrant Factor IX protein.

Embodiment 71. The method of any of Embodiments 62-69, wherein themammal has hemophilia B.

Embodiment 72. The method of any of Embodiments 62-69, wherein thenucleic acid sequence, expression vector, or virus vector is deliveredto the mammal intravenously, intraarterially, intramuscularly,subcutaneously, orally, by intubation, via catheter, dermally,intra-cranially, via inhalation, intra-cavity, or mucosally.

Embodiment 73. The method of any of Embodiments 62-69, wherein themammal is human.

Embodiment 74. The method of any of Embodiments 62-69, wherein themammal is sero-positive or sero-negative for an AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV-Rh74 serotype.

Embodiment 75. The method of any of Embodiments 62-69, furthercomprising administering empty capsid AAV.

Embodiment 76. The method of any of Embodiments 62-69, furthercomprising administering empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74 serotype.

Embodiment 77. The method of any of Embodiments 62-69, furthercomprising administering empty capsid AAV of the same serotype as theAAV vector administered.

Embodiment 78. The method of any of Embodiments 62-69, wherein saidFactor IX protein is expressed in the mammal at levels having atherapeutic effect on the mammal.

Embodiment 79. The method of any of Embodiments 62-69, wherein saidFactor IX protein is expressed in the mammal at levels having atherapeutic effect for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days, weeks or months.

Embodiment 80. The method of any of Embodiments 62-69, wherein saidFactor IX protein is present in the mammal at levels of about 20% FIXactivity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months.

Embodiment 81. The method of any of Embodiments 62-69, wherein the virusvector is administered at a dose in a range from about 1×10¹⁰-1×10¹¹,1×10¹¹-1×10¹², 1×10¹²-1×10¹³, or 1×10¹³-1×10¹⁴ vector genomes perkilogram (vg/kg) of the mammal.

Embodiment 82. The method of any of Embodiments 62-69, wherein the virusvector is administered at a dose of less than 1×10¹² vector genomes perkilogram (vg/kg) of the mammal, and said Factor IX protein is producedin the mammal at levels of about 20% activity or greater than 20%activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14continuous days, weeks or months

Embodiment 83. The method of any of Embodiments 62-69, wherein the virusvector is administered at a dose of about 5×10¹¹ vector genomes perkilogram (vg/kg) of the mammal, and said Factor IX protein is producedin the mammal at levels of about 20% activity or greater than 20%activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14continuous days, weeks or months.

Embodiment 84. The method of any of Embodiments 62-69, wherein saidnucleic acid sequence, expression vector, or virus vector administeredto the mammal does not produce a substantial immune response against theFactor IX protein and/or the virus vector.

Embodiment 85. The method of any of Embodiments 62-69, wherein asubstantial immune response against Factor IX protein and/or the virusvector is not produced for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 continuous days, weeks or months.

Embodiment 86. The method of any of Embodiments 62-69, wherein themammal does not produce a substantial immune response against the FactorIX protein.

Embodiment 87. The method of Embodiments 62-69, wherein said mammal doesnot develop an immune response against the Factor IX protein sufficientto decrease or block the Factor IX protein therapeutic effect.

Embodiment 88. The method of any of Embodiments 62-69, wherein themammal does not produce a substantial immune response against Factor IXprotein sufficient to decrease or block the Factor IX proteintherapeutic effect for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 continuous days, weeks or months.

Embodiment 89. The method of any of Embodiments 62-69, wherein saidmammal does not develop an immune response against the Factor IX proteinsufficient to decrease or block the Factor IX protein therapeutic effectfor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuousdays, weeks or months.—

Embodiment 90. The method of any of Embodiments 62-69, wherein saidmammal does not develop an immune response against AAV vector sufficientto decrease or block the Factor IX protein therapeutic effect.

Embodiment 91. The method of any of Embodiments 62-69, wherein saidmammal does not develop an immune response against AAV vector sufficientto decrease or block the Factor IX protein therapeutic effect for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days,weeks or months.

Embodiment 92. The method of any of Embodiments 62-69, wherein saidFactor IX protein is expressed in the mammal in amounts or at activitylevels having a therapeutic effect on the mammal without administeringan immunosuppressing agent (e.g., steroid).

Embodiment 93. The method of Embodiments any of Embodiments 62-69,wherein said Factor IX protein is expressed in the mammal in amounts orat activity levels having a therapeutic effect without administering animmunosuppressing agent (e.g., steroid) for at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 days, weeks or months.

Embodiment 94. The method of any of Embodiments 62-69, wherein saidmammal does not develop abnormally high levels of liver ALT, AST and/orLDH enzymes for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14continuous days, weeks or months.

Embodiment 95. The method of any of Embodiments 62-69, wherein saidmammal does not develop abnormally high levels of liver ALT, AST and/orLDH enzymes for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14continuous days, weeks or months to require using an immunosuppressingagent (e.g., steroid).

Embodiment 96. The method of any of Embodiments 62-69, wherein saidFactor IX protein is expressed in the mammal at levels greater than thecirculating levels of FIX needed to reduce the duration, severity orfrequency of spontaneous joint bleeds or cerebral bleeding for at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous weeks ormonths.

Embodiment 97. The method of any of Embodiments 62-69, wherein saidFactor IX protein is expressed in the mammal at levels for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks ormonths such that no recombinant FIX protein is needed or administeredafter AAV vector administration.

Embodiment 98. A recombinant AAV vector for treating hemophilia B,comprising a capsid and a genome, wherein said capsid comprises a VP1protein having the amino acid sequence of SEQ ID NO:4; and wherein saidgenome is single-stranded and comprises the following elements in 5′ to3′ order:

-   -   (a) a first AAV2 ITR,    -   (b) an ApoE HCR-1 enhancer,    -   (c) an AAT promoter,    -   (d) a codon-optimized nucleic acid encoding a human Factor IX        Padua, wherein the nucleic acid lacks at least one CpG        dinucleotide otherwise present,    -   (e) a polyadenylation sequence; and    -   (f) a second AAV2 ITR.

Embodiment 99. The rAAV vector of Embodiment 98, wherein

-   -   (a) the nucleic acid sequence of the first AAV2 ITR consists of        nucleotides 1-141 of SEQ ID NO:12,    -   (b) the nucleic acid sequence of said ApoE HCR-1 enhancer        consists of nucleotides 152-472 of SEQ ID NO:12,    -   (c) the nucleic acid sequence of said ATT promoter consists of        nucleotides 482-878 of SEQ ID NO:12,    -   (d) the nucleic acid sequence of said nucleic acid encoding        human Factor IX Padua variant consists of nucleotides 908-3731        of SEQ ID NO:12,    -   (e) the nucleic acid sequence of said polyadenylation sequence        consists of nucleotides 3820-4047 of SEQ ID NO:12; and    -   (f) the nucleic acid sequence of said second AAV2 ITR consists        of nucleotides 4097-4204 of SEQ ID NO:12.

Embodiment 100. The rAAV vector of Embodiment 99, wherein the genomecomprises a nucleic acid sequence corresponding to nucleotides 1-4204 ofSEQ ID NO:12.

Embodiment 101. A method for treating a human subject with hemophilia Bcomprising administering to said subject a therapeutically effectiveamount of the rAAV vector of any one of Embodiments 98 or 99.

Embodiment 102. The method of Embodiment 101, wherein said subject hassevere hemophilia B, and wherein said treatment is effective to reducethe hemophilia symptoms from severe to those of moderate or mildhemophilia B.

Embodiment 103. The method of Embodiment 101, wherein said treatment iseffective to achieve a level of plasma FIX activity that is at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, or 50% of normal FIX activity.

Embodiment 104. The method of Embodiment 103, wherein said treatment iseffective to achieve the level of plasma FIX activity for a sustainedperiod of at least 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months,15 months, 16 months, 17 months, 1.5 years, 2 years, 2.5 years, 3 years,3.5 years, 4 years, 4.5 years, or 5 years.

Embodiment 105. The method of Embodiment 104, wherein said treatment iseffective to achieve at least 1% plasma FIX activity for a sustainedperiod of at least 6 months, 1 year, 2 years, 3 years, 4 years, or 5years.

Embodiment 106. The method of Embodiment 104, wherein said treatment iseffective to achieve at least 5% plasma FIX activity for a sustainedperiod of at least 6 months, 1 year, 2 years, 3 years, 4 years, or 5years.

Embodiment 107. The method of Embodiment 104, wherein said treatment iseffective to achieve at least 10% plasma FIX activity for a sustainedperiod of at least 6 months, 1 year, 2 years, 3 years, 4 years, or 5years.

Embodiment 108. The method of Embodiment 104, wherein said treatment iseffective to achieve at least 20% plasma FIX activity for a sustainedperiod of at least 6 months, 1 year, 2 years, 3 years, 4 years, or 5years.

Embodiment 109. The method of Embodiment 104, wherein said treatment iseffective to achieve at least 30% plasma FIX activity for a sustainedperiod of at least 6 months, 1 year, 2 years, 3 years, 4 years, or 5years.

Embodiment 110. The method of Embodiment 104, wherein said treatment iseffective to achieve at least 40% plasma FIX activity for a sustainedperiod of at least 6 months, 1 year, 2 years, 3 years, 4 years, or 5years.

Embodiment 111. The method of Embodiment 104, wherein said treatment iseffective to achieve at least 20% plasma FIX activity for a sustainedperiod of at least 6 months.

Embodiment 112. The method of Embodiment 111, wherein thetherapeutically effective amount of said AAV vector is about 5.0×10¹¹vg/kg.

Embodiment 113. The method of Embodiment 101, wherein the treatment iseffective to reduce the frequency with which an average human subjecthaving severe hemophilia B requires FIX protein replacement therapy tomaintain adequate hemostasis by about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Embodiment 114. The method of Embodiment 101, wherein the treatment iseffective to reduce the frequency of spontaneous bleeding into thejoints of a human subject with severe hemophilia B by about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%, compared to the average untreated human subjectwith severe hemophilia B.

Embodiment 115. The method of Embodiment 103, wherein said AAV vectorresults in an antibody titer against the capsid that is not greater than1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, or 1:20, when determined at least 4 weeks after administration.

Embodiment 116. The method of Embodiment 103, wherein said AAV vectorresults in a T cell immune response against the capsid as measured usingan ELISPOT assay resulting in not more than 10, 20, 30, 40, 50, 100,200, 300, 400, or 500 spot-forming units per 1 million PBMCs whendetermined at least 4 weeks after administration.

Embodiment 117. The method of Embodiment 103, wherein said AAV vectorresults in an elevated liver enzyme level not more than 0%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%, ofthe upper limit of normal (ULN) value for the enzyme.

Embodiment 118. The method of Embodiment 117, wherein said enzyme isalanine aminotransferase (ALT), aspartate aminotransferase (AST), orlactate dehydrogenase (LDH).

Embodiment 119. The method of Embodiment 101, wherein said treatment iseffective to achieve a mean plasma FIX activity that is at least 1%, 5%,10%, 20%, 30%, or 40%, of normal, with a standard deviation less than15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, when measured atleast 8 weeks after the vector is administered.

Embodiment 120. The method of any one of Embodiments 101-119, whereinsaid AAV vector is administered in a pharmaceutical compositioncomprising empty capsids wherein said empty capsids comprise a VP1protein having the amino acid sequence of SEQ ID NO:4.

Embodiment 121. The method of Embodiment 120, wherein the ratio of saidempty capsids to said AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, or 10:1.

Embodiment 122. A host cell comprising a contiguous nucleic acidsequence identical to the nucleic acid sequence from 1-4204 of SEQ IDNO:12.

Embodiment 123. The host cell of Embodiment 122, further comprising AAVrep protein, AAV capsid protein and adenovirus helper protein(s).

Embodiment 124. The host cell of Embodiment 123, wherein the AAV capsidprotein comprising SEQ ID NO:4, or a sequence having 90% or moreidentity thereto.

Embodiment 125. The host cell of Embodiment 123, wherein the host cellexpresses FIX Padua protein.

Embodiment 126. A method of producing an AAV vector comprising thenucleic acid sequence from 1-4204 of SEQ ID NO:12, comprising culturinga host cell of Embodiment 123 under conditions allowing packing of thenucleic acid sequence from 1-4204 of SEQ ID NO:12 into AAV particlesthereby producing AAV vector.

Embodiment 127. The method of Embodiment 126, further comprisingpurifying or isolating the AAV vector so produced.

Embodiment 128. An AAV vector produced by the method of Embodiment 126.

Embodiment 129. An isolated or purified AAV vector produced by themethod of Embodiment 127.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of Rh74 VP1.

FIG. 2 shows the amino acid sequence or Rh74 VP2.

FIG. 3 shows the amino acid sequence of Rh74 VP3.

FIG. 4 shows the amino acid sequence of capsid variant 4-1 VP1 protein.

FIG. 5 shows the amino acid sequence of capsid variant 15-1.

FIG. 6 shows the amino acid sequence of capsid variant 15-2.

FIG. 7 shows the amino acid sequence of capsid variant 15-3/15-5.

FIG. 8 shows the amino acid sequence of capsid variant 15-4.

FIG. 9 shows the amino acid sequence of capsid variant 15-6.

FIG. 10 shows the nucleic acid sequence of FIX39.

FIG. 11 shows the nucleic acid sequence of FIX19.

FIG. 12A shows the sequence of the FIX39 plasmid.

FIG. 12B shows the sequence of the phFIX39v2 plasmid.

FIG. 13 shows a map of the FIX39 plasmid (SEQ ID NO:12).

FIG. 14 shows the Intron A nucleic acid sequence. According to certainembodiments, pharmaceutical compositions comprising AAV vectors includethose comprising the 4-1 capsid variant proteins (VP1, VP2 and VP3),comprise an excess of empty capsids greater than the concentration ofAAV vectors (i.e.,

FIG. 15 shows the nucleic acid sequence of FIX39+Intron A.

FIG. 16 shows transduction efficiency of the AAV-4-1 capsid variant (SEQID NO:4) analyzed in an in vitro setting.

FIG. 17 shows levels of hFIX in plasma of wild-type mice followingintravenous injection at week 8 of life with either 1×10¹¹ or 1×10¹²vg/kg of AAV-FIX39-Padua (square/circle) and AAV-FIX19-Padua(diamond/hexagon). Human FIX plasma levels were assayed by ELISA andrepresent multiple measurements, obtained by serial bleeding, on thesame group of animals during the course of the study (n=5 mice in eachcohort). Error bars denote standard error of the mean.

FIG. 18 shows circulating levels of human FIX in mouse plasma 24 hoursfollowing hydrodynamic tail vein injection of 5 μg of pFIX19-Padua orpFIX39-Padua plasmids. P=0.3337.

FIG. 19 shows a data summary of four human hemophilia B patientsadministered a single infusion of an AAV-FIX Padua variant (FIX39)bearing vector in accordance with the invention, and the FIX activity(%) over the ensuing evaluation periods (183, 102, 69 and 50 days,respectively).

FIG. 20A shows the FIX activity (%) data of the first human hemophilia Bpatient administered the single infusion of AAV-FIX Padua variant(FIX39) bearing vector, over the 183 day evaluation period.

FIG. 20B shows liver function test (ALT, AST and LDH enzymes) data ofthe first human hemophilia B patient administered the single infusion ofAAV-FIX Padua variant (FIX39) bearing vector, over the 183 dayevaluation period. The plotted LDH values (LDH¹) have been divided by 10in order to be shown with the ALT and AST values.

FIG. 21A shows the FIX activity (%) data of the second human hemophiliaB patient administered the single infusion of AAV-FIX Padua variant(FIX39) bearing vector, over the 102 day evaluation period.

FIG. 21B shows liver function test (ALT, AST and LDH enzymes) data ofthe second human hemophilia B patient administered the single infusionof AAV-FIX Padua variant (FIX39) bearing vector, over the 102 dayevaluation period. The plotted LDH values (LDH¹) have been divided by 10in order to be shown with the ALT and AST values.

FIG. 22A shows the FIX activity (%) data of the third human hemophilia Bpatient administered the single infusion of AAV-FIX Padua variant(FIX39) bearing vector, over the 69 day evaluation period.

FIG. 22B shows liver function test (ALT, AST and LDH enzymes) data ofthe third human hemophilia B patient administered the single infusion ofAAV-FIX Padua variant (FIX39) bearing vector, over the 69 day evaluationperiod. The plotted LDH values (LDH¹) have been divided by 10 in orderto be shown with the ALT and AST values.

FIG. 23A shows the FIX activity (%) data of the fourth human hemophiliaB patient administered the single infusion of AAV-FIX Padua variant(FIX39) bearing vector, over the 50 day evaluation period.

FIG. 23B shows liver function test (ALT, AST and LDH enzymes) data ofthe fourth human hemophilia B patient administered the single infusionof AAV-FIX Padua variant (FIX39) bearing vector, over the 50 dayevaluation period. The plotted LDH values (LDH¹) have been divided by 10in order to be shown with the ALT and AST values.

FIG. 24A shows low immunogenicity profile of AAV-FIX39-Padua in humansubjects.

FIG. 24B shows a comparative immunogenicity profile of AAV-FIX39-Paduaand AAV8-FIX19 in human subjects.

DETAILED DESCRIPTION

The invention is based, at least in part, on development of modifiednucleic acid sequences encoding proteins, such as human FIX protein. Invarious embodiments, a modified nucleic acid has a reduced number of CpG(cytosine-guanine) di-nucleotides compared to a reference nucleic acidsequence encoding Factor IX, such as a native (wild-type) sequenceencoding human Factor IX. In further embodiments, such modified nucleicacids having a reduced number of CpG di-nucleotides compared to areference Factor IX encoding nucleic acid (e.g., a native sequenceencoding human Factor IX) are included within expression vectors (e.g.,vector genomes) or plasmids.

The invention also includes compositions, such as compositions includinga modified nucleic acid sequence encoding human FIX. In suchcompositions, a modified nucleic acid has a reduced number of CpGdi-nucleotides relative to a reference sequence such as a native(wild-type) sequence encoding human Factor IX. Compositions also includeexpression vectors (e.g., viral vectors/vector genomes) and plasmidsthat include such modified nucleic acid sequences encoding human FIXprotein having a reduced number of CpG di-nucleotides.

In particular aspects, a nucleic acid sequence encoding human FIXprotein has 1-5 fewer CpG di-nucleotides than native sequence encodinghuman Factor IX; or has 5-10 fewer CpG di-nucleotides than native(wild-type) sequence encoding human Factor IX; or has 10-15 fewer CpGdi-nucleotides than native (wild-type) sequence encoding human FactorIX; or has 15-20 fewer CpG di-nucleotides than native (wild-type)sequence encoding human Factor IX; or has 20-25 fewer CpG di-nucleotidesthan native (wild-type) sequence encoding human Factor IX; or has 25-30fewer CpG di-nucleotides than native (wild-type) sequence encoding humanFactor IX; or has 30-40 fewer CpG di-nucleotides than native (wild-type)sequence encoding human Factor IX; or has 40-55 fewer CpG di-nucleotidesthan native (wild-type) sequence encoding human Factor IX; or iscompletely devoid of any CpG di-nucleotides.

Modified nucleic acids encoding Factor IX, such as FIX with a reducednumber of CpG dinucleotide, may further include one or more additionalcis elements. Representative cis elements include, without limitation,expression control elements, introns, ITRs, stop codons, polyAsequences, and/or filler polynucleotide sequences. In particularembodiments, such cis-acting elements can also be modified. For examplecis acting elements such as expression control elements, introns, ITRs,poly-A sequences, and/or filler polynucleotide sequences can have areduced number of CpG di-nucleotides. In one aspect, one or more cisacting elements, such as expression control elements, introns, ITRs,poly-A sequences, and/or filler polynucleotide sequences, are devoid ofCpG di-nucleotides. In particular aspects, one or more cis actingelements such as expression control elements, introns, ITRs, poly-Asequences, and/or filler polynucleotide sequences has 1-5 fewer CpGdi-nucleotides than a reference cis-acting element; or has 5-10 fewerCpG di-nucleotides than a reference cis-acting element; or has 10-15fewer CpG di-nucleotides than a reference cis-acting element; or has15-20 fewer CpG di-nucleotides than a reference cis-acting element; orhas 20-25 fewer CpG di-nucleotides than a reference cis-acting element;or has 25-30 fewer CpG di-nucleotides than a reference cis-actingelement; or has 30-40 fewer CpG di-nucleotides than a referencecis-acting element; or has 40-55 fewer CpG di-nucleotides than areference cis element; or is devoid of any CpG di-nucleotides.

The invention also includes viral vectors that include a modifiednucleic acid sequence encoding human FIX protein, such as FIX with areduced number of CpG dinucleotides. In particular embodiments, a vectorincludes a lenti- or parvo-viral vector, such as an adeno-viral vector.In a more particular embodiment, a modified nucleic acid sequenceencoding human FIX protein, such as FIX with a reduced number of CpGdinucleotides, is comprised in an adeno-associated virus (AAV) vector.

In further particular embodiments, adeno-associated virus (AAV) vectorsinclude capsids derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8, as well as variants(e.g., capsid variants, such as amino acid insertions, additions andsubstitutions) thereof. As will be appreciated by one of ordinary skillin the art, AAV capsids typically include a VP1 protein and two shorterproteins, called VP2 and VP3, that are essentially amino-terminaltruncations of VP1. Depending on the capsid and other factors known tothose of ordinary skill, the three capsid proteins VP1, VP2 and VP3 aretypically present in a capsid at a ratio approximating 1:1:10,respectively, although this ratio, particularly of VP3, can varysignificantly and should not to be considered limiting in any respect.

AAV variants include AAV-Rh74 variants, for example AAV capsid variantsof the Rh74 VP1 capsid sequence (SEQ ID NO:1; FIG. 1), including but notlimited to variants 4-1, 15-1, 15-2, 15-3/15-5, 15-4 and 15-6 describedin Table 1. Rh74 VP2 and Rh74 VP3 amino acid sequences are provided inSEQ ID NO:2 (FIG. 2) and SEQ ID NO:3 (FIG. 3), respectively.

TABLE 1 AAV capsid variants Amino Acid Substitutions and IndicatedPositions in Sequence Variant Rh74 VP1 Capsid Identifier FIG.  4-1G195A-L199V- S201P-G202N SEQ ID NO: 4 FIG. 4 15-1G195A-L199V-S201P-G202N SEQ ID NO: 5 FIG. 5 K(137/259/333/530/552/569/38/51/77/169/547)R 15-2 G195A-L199V- S201P-G202N SEQ ID NO: 6 FIG. 6K(137/259/333/530/552/ 569/38/51/77/163/169)R 15-3/15-5 G195A-L199V-S201P-G202N SEQ ID NO: 7 FIG. 7 K(137/259/333/530/552/569/38/51/77/163/547)R (variant 15-3) G195A-L199V- S201P-G202NK(137/259/333/530/552/ 569/38/51/77/547/163)R (variant 15-5) 15-4G195A-L199V- S201P-G202N SEQ ID NO: 8 FIG. 8 K(137/259/333/530/552/569/38/51/77/163/668)R 15-6 G195A-L199V- S201P-G202N SEQ ID NO: 9 FIG. 9K(137/259/333/530/552/ 569/38/51/77/547/688)R

4-1 variant (SEQ ID NO:4) had an alanine, a valine, a proline, and anasparagine substitution at amino acid positions 195, 199, 201 and 202,respectively, of VP1 capsid. The 4-1 variant VP1 capsid amino acidsequence, with substituted residues a, v, p and n, underlined and inbold is shown in FIG. 4 (SEQ ID NO:4). For variant 4-1, the VP2 sequenceconsists of SEQ ID NO:27, and the VP3 sequence consists of SEQ ID NO:3,respectively.

15-1, 15-2, 15-3, 15-4, 1-5 and 15-6 variants also had an alanine, avaline, a proline, and an asparagine substitution at amino acidpositions 195, 199, 201 and 202, respectively, of VP1 capsid. Inaddition, these variants had multiple arginine substitutions of lysineat various positions. The 15-1 variant VP1 capsid amino acid sequence(SEQ ID NO:5) is shown in FIG. 5; the 15-2 variant VP1 capsid amino acidsequence (SEQ ID NO:6) is shown in FIG. 6; the 15-3/15-5 variant VP1capsid amino acid sequence (SEQ ID NO:7) is shown in FIG. 7; the 15-4variant VP1 capsid amino acid sequence (SEQ ID NO:8) is shown in FIG. 8;and the 15-6 variant VP1 capsid amino acid sequence (SEQ ID NO:9) isshown in FIG. 9. Examples of capsids that may be used herein include,but are not limited to, those described in United States patentpublication no. 2015/0023924.

Accordingly, lenti- and parvo-viral vectors such as AAV vectors andviral vector variants such as AAV variants (e.g., capsid variants suchas 4-1, 15-1, 15-2, 15-3/15-5, 15-4 and 15-6) that include (encapsidateor package) vector genome including modified nucleic acid sequenceencoding human FIX protein, such as FIX with a reduced number of CpGdinucleotides, are provided.

In exemplary studies, AAV-Rh74 mediated gene transfer/delivery producedprotein expression levels that were significantly higher than severalother serotypes. In particular, AAV-Rh74 vector and capsid variants(e.g., 4-1) target genes for delivery to the liver with efficiency atleast comparable to the gold standard for liver transduction, AAV8, inhemophilia B dogs and/or in mice and/or macaques.

As set forth herein, viral vectors such as lenti- and parvo-virusvectors, including AAV serotypes and variants provide a means fordelivery of polynucleotide sequences into cells ex vivo, in vitro and invivo, which can encode proteins such that the cells express the encodedproteins. For example, a recombinant AAV vector can include aheterologous polynucleotide encoding a desired protein or peptide (e.g.,Factor IX). Vector delivery or administration to a subject (e.g.,mammal) therefore provides encoded proteins and peptides to the subject.Thus, viral vectors such as lenti- and parvo-virus vectors, includingAAV serotypes and variants such as capsid variants (e.g., 4-1) can beused to transfer/deliver heterologous polynucleotides for expression,and optionally for treating a variety of diseases.

In particular embodiments, a recombinant vector (e.g., AAV) is aparvovirus vector. Parvoviruses are small viruses with a single-strandedDNA genome. “Adeno-associated viruses” (AAV) are in the parvovirusfamily.

Parvoviruses including AAV are viruses useful as gene therapy vectors asthey can penetrate cells and introduce nucleic acid/genetic material sothat the nucleic acid/genetic material may be stably maintained incells. In addition, these viruses can introduce nucleic acid/geneticmaterial into specific sites, for example, such as a specific site onchromosome 19. Because AAV are not associated with pathogenic disease inhumans, AAV vectors are able to deliver heterologous polynucleotidesequences (e.g., therapeutic proteins and agents) to human patientswithout causing substantial AAV pathogenesis or disease.

AAV and AAV variants (e.g., capsid variants such as 4-1) serotypes(e.g., VP1, VP2, and/or VP3 sequences) may or may not be distinct fromother AAV serotypes, including, for example, AAV1-AAV11, Rh74 or Rh10(e.g., distinct from VP1, VP2, and/or VP3 sequences of any ofAAV1-AAV11, Rh74 or Rh10 serotypes).

As used herein, the term “serotype” is a distinction used to refer to anAAV having a capsid that is serologically distinct from other AAVserotypes. Serologic distinctiveness is determined on the basis of thelack of cross-reactivity between antibodies to one AAV as compared toanother AAV. Such cross-reactivity differences are usually due todifferences in capsid protein sequences/antigenic determinants (e.g.,due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).Despite the possibility that AAV variants including capsid variants maynot be serologically distinct from a reference AAV or other AAVserotype, they differ by at least one nucleotide or amino acid residuecompared to the reference or other AAV serotype.

Under the traditional definition, a serotype means that the virus ofinterest has been tested against serum specific for all existing andcharacterized serotypes for neutralizing activity and no antibodies havebeen found that neutralize the virus of interest. As more naturallyoccurring virus isolates of are discovered and/or capsid mutantsgenerated, there may or may not be serological differences with any ofthe currently existing serotypes. Thus, in cases where the new virus(e.g., AAV) has no serological difference, this new virus (e.g., AAV)would be a subgroup or variant of the corresponding serotype. In manycases, serology testing for neutralizing activity has yet to beperformed on mutant viruses with capsid sequence modifications todetermine if they are of another serotype according to the traditionaldefinition of serotype. Accordingly, for the sake of convenience and toavoid repetition, the term “serotype” broadly refers to bothserologically distinct viruses (e.g., AAV) as well as viruses (e.g.,AAV) that are not serologically distinct that may be within a subgroupor a variant of a given serotype.

Recombinant vector (e.g., AAV) plasmids, vector (e.g., AAV) genomes, aswell as methods and uses thereof, include any viral strain or serotype.As a non-limiting example, a recombinant vector (e.g., AAV) plasmid orvector (e.g., AAV) genome can be based upon any AAV genome, such asAAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -rh74, -rh10 orAAV-2i8, for example. Such vectors can be based on the same of strain orserotype (or subgroup or variant), or be different from each other. As anon-limiting example, a recombinant vector (e.g., AAV) plasmid or vector(e.g., AAV) genome based upon one serotype genome can be identical toone or more of the capsid proteins that package the vector. In addition,a recombinant vector (e.g., AAV) plasmid or vector (e.g., AAV) genomecan be based upon an AAV (e.g., AAV2) serotype genome distinct from oneor more of the capsid proteins that package the vector, in which case atleast one of the three capsid proteins could be a AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8or variant such as AAV-Rh74 variant (e.g., capsid variants such as 4-1,15-1, 15-2, 15-3/15-5, 15-4 and 15-6), for example.

AAV vectors therefore include gene/protein sequences identical togene/protein sequences characteristic for a particular serotype. As usedherein, an “AAV vector related to AAV1” refers to one or more AAVproteins (e.g., VP1, VP2, and/or VP3 sequences) that has substantialsequence identity to one or more polynucleotides or polypeptidesequences that comprise AAV1. Analogously, an “AAV vector related toAAV8” refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3sequences) that has substantial sequence identity to one or morepolynucleotides or polypeptide sequences that comprise AAV8. An “AAVvector related to AAV-Rh74” refers to one or more AAV proteins (e.g.,VP1, VP2, and/or VP3 sequences) that has substantial sequence identityto one or more polynucleotides or polypeptide sequences that compriseAAV-Rh74. (see, e.g., VP1, VP2, VP3 of FIGS. 1-3). Such AAV vectorsrelated to another serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, can thereforehave one or more distinct sequences from AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8, but canexhibit substantial sequence identity to one or more genes and/orproteins, and/or have one or more functional characteristics of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74or AAV-2i8 (e.g., such as cell/tissue tropism). Exemplary non-limitingAAV-Rh74 and related AAV variants such as AAV-Rh74 or related AAV suchas AAV-Rh74 variants (e.g., capsid variants such as 4-1, 15-1, 15-2,15-3/15-5, 15-4 and 15-6) sequences include VP1, VP2, and/or VP3 setforth herein, for example, in FIGS. 1-9.

In various exemplary embodiments, an AAV vector related to a referenceserotype has a polynucleotide, polypeptide or subsequence thereof thatincludes or consists of a sequence at least 80% or more (e.g., 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74or AAV-2i8 (e.g., such as AAV-Rh74 VP1, VP2, and/or VP3 sequences setforth in FIGS. 1-9).

Methods and uses of the invention include AAV sequences (polypeptidesand nucleotides), AAV-Rh74 sequences (polypeptides and nucleotides) andsubsequences thereof that exhibit less than 100% sequence identity to areference AAV serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, Rh10, or AAV-2i8, for example, AAV-Rh74 geneor protein sequence (e.g., VP1, VP2, and/or VP3 sequences set forth inFIGS. 1-9), but are distinct from and not identical to known AAV genesor proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, genes or proteins, etc. Inone embodiment, an AAV polypeptide or subsequence thereof includes orconsists of a sequence at least 80% or more identical, e.g., 85%, 85%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,etc., i.e. up to 100% identical to any reference AAV sequence orsubsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 (e.g., VP1, VP2 and/orVP3 sequences set forth in FIGS. 1-9). In particular aspects, an AAVvariant has one, two, three or four of the four amino acid substitutions(e.g., capsid variant 4-1, 15-1, 15-2, 15-3/15-5, 15-4 and 15-6).

Recombinant vectors (e.g., AAV), including AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 and variant,related, hybrid and chimeric sequences, can be constructed usingrecombinant techniques that are known to the skilled artisan, to includeone or more heterologous polynucleotide sequences (transgenes) flankedwith one or more functional AAV ITR sequences. Such vectors can have oneor more of the wild type AAV genes deleted in whole or in part, forexample, a rep and/or cap gene, but retain at least one functionalflanking ITR sequence, as necessary for the rescue, replication, andpackaging of the recombinant vector into an AAV vector particle. An AAVvector genome would therefore include sequences required in cis forreplication and packaging (e.g., functional ITR sequences)

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein to refer to all forms of nucleic acid, oligonucleotides,including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).Polynucleotides include genomic DNA, cDNA and antisense DNA, and splicedor unspliced mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, e.g.,small or short hairpin (sh)RNA, microRNA (miRNA), small or shortinterfering (si)RNA, trans-splicing RNA, or antisense RNA).Polynucleotides include naturally occurring, synthetic, andintentionally modified or altered polynucleotides (e.g., having reducedCpG dinucleotides). Polynucleotides can be single, double, or triplex,linear or circular, and can be of any length. In discussingpolynucleotides, a sequence or structure of a particular polynucleotidemay be described herein according to the convention of providing thesequence in the 5′ to 3′ direction.

A “heterologous” polynucleotide refers to a polynucleotide inserted intoa vector (e.g., AAV) for purposes of vector mediated transfer/deliveryof the polynucleotide into a cell. Heterologous polynucleotides aretypically distinct from vector (e.g., AAV) nucleic acid, i.e., arenon-native with respect to viral (e.g., AAV) nucleic acid. Oncetransferred/delivered into the cell, a heterologous polynucleotide,contained within the vector, can be expressed (e.g., transcribed, andtranslated if appropriate). Alternatively, a transferred/deliveredheterologous polynucleotide in a cell, contained within the vector, neednot be expressed. Although the term “heterologous” is not always usedherein in reference to polynucleotides, reference to a polynucleotideeven in the absence of the modifier “heterologous” is intended toinclude heterologous polynucleotides in spite of the omission. Anexample of a heterologous sequence would be a Factor IX encoding nucleicacid, for example, a modified nucleic acid encoding Factor IX, such as anucleic acid having reduced CpG dinucleotides relative to a referencenucleic acid sequence.

The “polypeptides,” “proteins” and “peptides” encoded by the“polynucleotide sequences,” include full-length native sequences, aswith naturally occurring proteins, as well as functional subsequences,modified forms or sequence variants so long as the subsequence, modifiedform or variant retains some degree of functionality of the nativefull-length protein. In methods and uses of the invention, suchpolypeptides, proteins and peptides encoded by the polynucleotidesequences can be but are not required to be identical to the endogenousprotein that is defective, or whose expression is insufficient, ordeficient in the treated mammal.

In the invention lenti- and parvo-viral vectors, such as an adeno-viralvector and AAV vectors, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 and related AAVvariants such as AAV-Rh74 variants (e.g., capsid variants such as 4-1,15-1, 15-2, 15-3/15-5, 15-4 and 15-6) can be used to introduce/deliverpolynucleotides stably or transiently into cells and progeny thereof.The term “transgene” is used herein to conveniently refer to such aheterologous polynucleotide that is intended or has been introduced intoa cell or organism. Transgenes include any polynucleotide, such as agene that encodes a polypeptide or protein (e.g., Factor IX).

For example, in a cell having a transgene, the transgene has beenintroduced/transferred by way of vector, such as AAV, “transduction” or“transfection” of the cell. The terms “transduce” and “transfect” referto introduction of a molecule such as a polynucleotide into a cell orhost organism.

A cell into which the transgene has been introduced is referred to as a“transduced cell.” Accordingly, a “transduced” cell (e.g., in a mammal,such as a cell or tissue or organ cell), means a genetic change in acell following incorporation of an exogenous molecule, for example, apolynucleotide or protein (e.g., a transgene) into the cell. Thus, a“transduced” cell is a cell into which, or a progeny thereof in which anexogenous molecule has been introduced, for example. The cell(s) can bepropagated and the introduced protein expressed, or nucleic acidtranscribed. For gene therapy uses and methods, a transduced cell can bein a subject.

The introduced polynucleotide may or may not be integrated into nucleicacid of the recipient cell or organism. If an introduced polynucleotidebecomes integrated into the nucleic acid (genomic DNA) of the recipientcell or organism it can be stably maintained in that cell or organismand further passed on to or inherited by progeny cells or organisms ofthe recipient cell or organism. Finally, the introduced nucleic acid mayexist in the recipient cell or host organism only transiently.

Cells that may be transduced include a cell of any tissue or organ type,of any origin (e.g., mesoderm, ectoderm or endoderm). Non-limitingexamples of cells include liver (e.g., hepatocytes, sinusoidalendothelial cells), pancreas (e.g., beta islet cells), lung, central orperipheral nervous system, such as brain (e.g., neural, glial orependymal cells) or spine, kidney, eye (e.g., retinal, cell components),spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscleor psoas, or gut (e.g., endocrine), adipose tissue (white, brown orbeige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes,osteoclasts, epithelial cells, endothelial cells, salivary gland cells,inner ear nervous cells or hematopoietic (e.g., blood or lymph) cells.Additional examples include stem cells, such as pluripotent ormultipotent progenitor cells that develop or differentiate into liver(e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., betaislet cells), lung, central or peripheral nervous system, such as brain(e.g., neural, glial or ependymal cells) or spine, kidney, eye (retinal,cell components), spleen, skin, thymus, testes, lung, diaphragm, heart(cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue(white, brown or beige), muscle (e.g., fibroblasts), synoviocytes,chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivarygland cells, inner ear nervous cells or hematopoietic (e.g., blood orlymph) cells.

A “therapeutic molecule” in one embodiment is a peptide or protein thatmay alleviate or reduce symptoms that result from an absence or defectin a protein in a cell or subject. Alternatively, a “therapeutic”peptide or protein encoded by a transgene is one that confers a benefitto a subject, e.g., to correct a genetic defect, to correct a gene(expression or functional) deficiency.

Non-limiting examples of heterologous polynucleotides encoding geneproducts (e.g., therapeutic proteins) which are useful in accordancewith the invention include those that may be used in the treatment of adisease or disorder including, but not limited to, blood clottingdisorders such as hemophilia A, hemophilia B, thalassemia, and anemia.

All mammalian and non-mammalian forms of polynucleotides encoding geneproducts, including the non-limiting genes and proteins disclosed hereinare expressly included, either known or unknown. Thus, the inventionincludes genes and proteins from non-mammals, mammals other than humans,and humans, which genes and proteins function in a substantially similarmanner to the human genes and proteins described herein. Non-limitingexamples of mammalian non-human Factor IX sequences are described inYoshitake et al., 1985, supra; Kurachi et al., 1995, supra; Jallat etal., 1990, supra; Kurachi et al., 1982, Proc. Natl. Acad. Sci. USA79:6461-6464; Jaye et al., 1983, Nucl. Acids Res. 11:2325-2335; Anson etal., 1984, EMBO J. 3: 1053-1060; Wu et al., 1990, Gene 86:275-278; Evanset al., Proc Natl Acad Sci USA 86:10095 (1989), Blood 74:207-212;Pendurthi et al., 1992, Thromb. Res. 65:177-186; Sakar et al., 1990,Genomics 1990, 6:133-143; and, Katayama et al., 1979, Proc. Natl. Acad.Sci. USA 76:4990-4994.

Polynucleotides, polypeptides and subsequences thereof include modifiedand variant forms. As used herein, the terms “modify” or “variant” andgrammatical variations thereof, mean that a polynucleotide, polypeptideor subsequence thereof deviates from a reference sequence. Modified andvariant sequences may therefore have substantially the same, greater orless activity or function than a reference sequence, but at least retainpartial activity or function of the reference sequence. In particularembodiments, a modified nucleic acid encodes Factor IX, and has beenmodified to reduce the number of CpG dinucleotides compared to areference Factor IX encoding nucleic acid (e.g., wild-type Factor IXsequence, such as a human or other mammalian Factor IX gene sequence).

Variants also include gain and loss of function variants. For example,wild type human Factor IX DNA sequences, which protein variants ormutants retain activity, or are therapeutically effective, or arecomparably or even more therapeutically active than invariant humanFactor IX in the methods and uses of the invention. In one non-limitingexample of a naturally occurring human Factor IX variant, called the“Padua”, human Factor IX has a L (leucine) at position 338 instead of anR (arginine). The Padua FIX has greater catalytic and coagulant activitycompared to human Factor IX lacking the Padua mutation. Changing Residue338 in Human Factor IX from Arginine to Alanine Causes an Increase inCatalytic Activity (Chang et al., J. Biol. Chem., 273:12089-94 (1998)).In another particular example, collagen IV serves to trap Factor IX,meaning that when introduced into the muscle tissue of a mammal some ofthe Factor IX is not available for participation in blood coagulationbecause it is retained in the interstitial spaces in the muscle tissue.A mutation in the sequence of Factor IX that results in a protein withreduced binding to collagen IV (e.g., loss of function) is a usefulmutant, for example, for treatment of hemophilia. An example of such amutant Factor IX gene encodes a human FIX protein with the amino acidalanine in place of lysine in the fifth amino acid position from thebeginning of the mature protein.

Non-limiting examples of modifications include one or more nucleotide oramino acid substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25,25-30, 30-40, 40-50, 50-100, or more nucleotides or residues), such assubstituting a CpG for an alternative dinucleotide in a transgene (e.g.,a Factor IX encoding gene, such as FIX encoding gene with a reducednumber of CpG dinucleotides). An example of an amino acid substitutionis a conservative amino acid substitution in a capsid sequence. Anotherexample of an amino acid substitution is an arginine for a lysineresidue (e.g., one or more arginine substitution of a lysine as setforth in any of 4-1, 15-1, 15-2, 15-3/15-5, 15-4 and/or 15-6). Furthermodifications include additions (e.g., insertions or 1-3, 3-5, 5-10,10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, or more nucleotides orresidues) and deletions (e.g., subsequences or fragments) of a referencesequence. In particular embodiments, a modified or variant sequenceretains at least part of a function or an activity of unmodifiedsequence. Such modified forms and variants can have the same, less than,or greater, but at least a part of, a function or activity of areference sequence, for example, as described herein.

As set forth herein, a variant can have one or more non-conservative ora conservative amino acid sequence differences or modifications, orboth. A “conservative substitution” is the replacement of one amino acidby a biologically, chemically or structurally similar residue.Biologically similar means that the substitution does not destroy abiological activity. Structurally similar means that the amino acidshave side chains with similar length, such as alanine, glycine andserine, or a similar size. Chemical similarity means that the residueshave the same charge or are both hydrophilic or hydrophobic. Particularexamples include the substitution of one hydrophobic residue, such asisoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acids, or glutamine forasparagine, serine for threonine, and the like. Particular examples ofconservative substitutions include the substitution of a hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,the substitution of a polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. For example, conservative aminoacid substitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. A “conservative substitution”also includes the use of a substituted amino acid in place of anunsubstituted parent amino acid.

Accordingly, the invention includes gene and protein variants (e.g., ofpolynucleotides encoding proteins described herein) which retain one ormore biological activities (e.g., function in blood clotting, etc.).Variants can differ from a reference sequence, such as naturallyoccurring polynucleotides, proteins or peptides. Such variants ofpolynucleotides, proteins or polypeptides include proteins orpolypeptides which have been or may be modified using recombinant DNAtechnology such that the polynucleotide, protein or polypeptidepossesses altered or additional properties.

At the nucleotide sequence level, a naturally and non-naturallyoccurring variant gene will typically be at least about 50% identical,more typically about 70% identical, even more typically about 80%identical to the reference gene. Thus, for example, a FIX gene with areduced number of CpG dinucleotides may have 80% or more identity towild-type FIX gene, or 80-85%, 85-90%, 90-95%, or more identity towild-type FIX gene, e.g., 96%, 97%, 98%, or 99% or more identity towild-type FIX gene.

At the amino acid sequence level, a naturally and non-naturallyoccurring variant protein will typically be at least about 70%identical, more typically about 80% identical, even more typically about90% or more identity to the reference protein, although substantialregions of non-identity are permitted in non-conserved regions (e.g.,less, than 70% identical, such as less than 60%, 50% or even 40%). Inother embodiments, the sequences have at least 60%, 70%, 75% or moreidentity (e.g., 80%, 85% 90%, 95%, 96%, 97%, 98%, 99% or more identity)to a reference sequence.

The term “identity,” “homology” and grammatical variations thereof, meanthat two or more referenced entities are the same, when they are“aligned” sequences. Thus, by way of example, when two polypeptidesequences are identical, they have the same amino acid sequence, atleast within the referenced region or portion. Where two polynucleotidesequences are identical, they have the same polynucleotide sequence, atleast within the referenced region or portion. The identity can be overa defined area (region or domain) of the sequence. An “area” or “region”of identity refers to a portion of two or more referenced entities thatare the same. Thus, where two protein or nucleic acid sequences areidentical over one or more sequence areas or regions they share identitywithin that region. An “aligned” sequence refers to multiplepolynucleotide or protein (amino acid) sequences, often containingcorrections for missing or additional bases or amino acids (gaps) ascompared to a reference sequence

The identity can extend over the entire length or a portion of thesequence. In particular aspects, the length of the sequence sharing thepercent identity is 2, 3, 4, 5 or more contiguous polynucleotide oramino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, etc. contiguous polynucleotides or amino acids. In additionalparticular aspects, the length of the sequence sharing identity is 21 ormore contiguous polynucleotide or amino acids, e.g., 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc.contiguous polynucleotides or amino acids. In further particularaspects, the length of the sequence sharing identity is 41 or morecontiguous polynucleotide or amino acids, e.g. 42, 43, 44, 45, 45, 47,48, 49, 50, etc., contiguous polynucleotides or amino acids. In yetfurther particular aspects, the length of the sequence sharing identityis 50 or more contiguous polynucleotide or amino acids, e.g., 50-55,55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110,etc. contiguous polynucleotide or amino acids.

The terms “homologous” or “homology” mean that two or more referencedentities share at least partial identity over a given region or portion.“Areas, regions or domains” of homology or identity mean that a portionof two or more referenced entities share homology or are the same. Thus,where two sequences are identical over one or more sequence regions theyshare identity in these regions. “Substantial homology” means that amolecule is structurally or functionally conserved such that it has oris predicted to have at least partial structure or function of one ormore of the structures or functions (e.g., a biological function oractivity) of the reference molecule, or relevant/corresponding region orportion of the reference molecule to which it shares homology.

The extent of identity (homology) between two sequences can beascertained using a computer program and/or mathematical algorithm. Suchalgorithms that calculate percent sequence identity (homology) generallyaccount for sequence gaps and mismatches over the comparison region orarea. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see,e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly availablethrough NCBI) has exemplary search parameters as follows: Mismatch-2;gap open 5; gap extension 2. For polypeptide sequence comparisons, aBLASTP algorithm is typically used in combination with a scoring matrix,such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 andFASTA3) and SSEARCH sequence comparison programs are also used toquantitate extent of identity (Pearson et al., Proc. Natl. Acad. Sci.USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smithet al., J. Mol. Biol. 147:195 (1981)). Programs for quantitating proteinstructural similarity using Delaunay-based topological mapping have alsobeen developed (Bostick et al., Biochem Biophys Res Commun. 304:320(2003)).

Polynucleotides include additions and insertions, for example, one ormore heterologous domains. An addition (e.g., heterologous domain) canbe a covalent or non-covalent attachment of any type of molecule to acomposition. Typically additions and insertions (e.g., a heterologousdomain) confer a complementary or a distinct function or activity.

Additions and insertions include chimeric and fusion sequences, which isa polynucleotide or protein sequence having one or more molecules notnormally present in a reference native (wild type) sequence covalentlyattached to the sequence. The terms “fusion” or “chimeric” andgrammatical variations thereof, when used in reference to a moleculemeans that a portions or part of the molecule contains a differententity distinct (heterologous) from the molecule—as they do nottypically exist together in nature. That is, for example, one portion ofthe fusion or chimera, includes or consists of a portion that does notexist together in nature, and is structurally distinct.

The term “vector” refers to a plasmid, virus (e.g., AAV vector), orother vehicle that can be manipulated by insertion or incorporation of apolynucleotide. Such vectors can be used for genetic manipulation (i.e.,“cloning vectors”), to introduce/transfer polynucleotides into cells,and to transcribe or translate the inserted polynucleotide in cells. Avector nucleic acid sequence generally contains at least an origin ofreplication for propagation in a cell and optionally additionalelements, such as a heterologous polynucleotide sequence, expressioncontrol element (e.g., a promoter, enhancer), intron, ITR(s), selectablemarker (e.g., antibiotic resistance), poly-Adenine (also referred to aspoly-adenylation) sequence.

A viral vector is derived from or based upon one or more nucleic acidelements that comprise a viral genome. Particular viral vectors includelenti- and parvo-virus vectors, such as adeno-associated virus (AAV)vectors.

As used herein, the term “recombinant,” as a modifier of viral vector,such as recombinant lenti- or parvo-virus (e.g., AAV) vectors, as wellas a modifier of sequences such as recombinant polynucleotides andpolypeptides, means that the compositions (e.g., AAV or sequences) havebeen manipulated (i.e., engineered) in a fashion that generally does notoccur in nature. A particular example of a recombinant vector, such asan AAV vector would be where a polynucleotide that is not normallypresent in the wild-type viral (e.g., AAV) genome is inserted within theviral genome. For example, an example of a recombinant polynucleotidewould be where a heterologous polynucleotide (e.g., gene) encoding aprotein is cloned into a vector, with or without 5′, 3′ and/or intronregions that the gene is normally associated within the viral (e.g.,AAV) genome. Although the term “recombinant” is not always used hereinin reference to vectors, such as viral and AAV vectors, as well assequences such as polynucleotides and polypeptides, recombinant forms ofviral, AAV, and sequences including polynucleotides and polypeptides,are expressly included in spite of any such omission.

A recombinant viral “vector” or “AAV vector” is derived from the wildtype genome of a virus, such as AAV by using molecular methods to removethe wild type genome from the virus (e.g., AAV), and replacing with anon-native nucleic acid, such as a heterologous polynucleotide sequence(e.g., modified nucleic acid sequence encoding human FIX, such as FIXwith a reduced number of CpG dinucleotides). Typically, for AAV one orboth inverted terminal repeat (ITR) sequences of AAV genome are retainedin the AAV vector. A “recombinant” viral vector (e.g., AAV) isdistinguished from a viral (e.g., AAV) genome, since all or a part ofthe viral genome has been replaced with a non-native sequence withrespect to the viral (e.g., AAV) genomic nucleic acid such as aheterologous polynucleotide sequence (e.g., modified nucleic acidsequence encoding human FIX, such as FIX with a reduced number of CpGdinucleotides). Incorporation of a non-native sequence (e.g., modifiednucleic acid sequence encoding human FIX, such as FIX with a reducednumber of CpG dinucleotides) therefore defines the viral vector (e.g.,AAV) as a “recombinant” vector, which in the case of AAV can be referredto as a “rAAV vector.”

A recombinant vector (e.g., lenti-, parvo-, AAV) sequence can bepackaged—referred to herein as a “particle” for subsequent infection(transduction) of a cell, ex vivo, in vitro or in vivo. Where arecombinant vector sequence is encapsidated or packaged into an AAVparticle, the particle can also be referred to as a “rAAV.” Suchparticles include proteins that encapsidate or package the vectorgenome. Particular examples include viral envelope proteins, and in thecase of AAV, capsid proteins.

For a recombinant plasmid, a vector “genome” refers to the portion ofthe recombinant plasmid sequence that is ultimately packaged orencapsidated to form a viral (e.g., AAV) particle. In cases whererecombinant plasmids are used to construct or manufacture recombinantvectors, the vector genome does not include the portion of the “plasmid”that does not correspond to the vector genome sequence of therecombinant plasmid. This non vector genome portion of the recombinantplasmid is referred to as the “plasmid backbone,” which is important forcloning and amplification of the plasmid, a process that is needed forpropagation and recombinant virus production, but is not itself packagedor encapsidated into virus (e.g., AAV) particles.

Thus, a vector “genome” refers to the portion of the vector plasmid thatis packaged or encapsidated by virus (e.g., AAV), and which contains aheterologous polynucleotide sequence. The non vector genome portion ofthe recombinant plasmid is the “plasmid backbone” that is important forcloning and amplification of the plasmid, e.g., has a selectable marker,such as Kanamycin, but is not itself packaged or encapsidated by virus(e.g., AAV).

Amounts of rAAV that encapsidate/package vector genomes can bedetermined, for example, by quantitative PCR. This assay measures thephysical number of packaged vector genomes by real-time quantitativepolymerase chain reaction and can be performed at various stages of themanufacturing/purification process, for example, on bulk AAV vector andfinal product.

Recombinant vector sequences are manipulated by insertion orincorporation of a polynucleotide. As disclosed herein, a vector plasmidgenerally contains at least an origin of replication for propagation ina cell and one or more expression control elements.

Vector sequences including AAV vectors can include one or more“expression control elements.” Typically, expression control elementsare nucleic acid sequence(s) that influence expression of an operablylinked polynucleotide. Control elements, including expression controlelements as set forth herein such as promoters and enhancers, presentwithin a vector are included to facilitate proper heterologouspolynucleotide transcription and if appropriate translation (e.g., apromoter, enhancer, splicing signal for introns, maintenance of thecorrect reading frame of the gene to permit in-frame translation of mRNAand, stop codons etc.). Such elements typically act in cis, referred toas a “cis acting” element, but may also act in trans.

Expression control can be effected at the level of transcription,translation, splicing, message stability, etc. Typically, an expressioncontrol element that modulates transcription is juxtaposed near the 5′end (i.e., “upstream”) of a transcribed polynucleotide (e.g., of amodified nucleic acid encoding Factor IX, such as FIX with a reducednumber of CpG dinucleotides). Expression control elements can also belocated at the 3′ end (i.e., “downstream”) of the transcribed sequenceor within the transcript (e.g., in an intron). Expression controlelements can be located adjacent to or at a distance away from thetranscribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500, ormore nucleotides from the polynucleotide), even at considerabledistances. Nevertheless, owing to the polynucleotide length limitationsof certain vectors, such as AAV vectors, such expression controlelements will typically be within 1 to 1000 nucleotides from thetranscribed polynucleotide.

Functionally, expression of operably linked heterologous polynucleotideis at least in part controllable by the element (e.g., promoter) suchthat the element modulates transcription of the polynucleotide and, asappropriate, translation of the transcript. A specific example of anexpression control element is a promoter, which is usually located 5′ ofthe transcribed sequence. Another example of an expression controlelement is an enhancer, which can be located 5′, 3′ of the transcribedsequence, or within the transcribed sequence.

A “promoter” as used herein can refer to a nucleic acid (e.g., DNA)sequence that is located adjacent to a polynucleotide sequence thatencodes a recombinant product. A promoter is typically operativelylinked to an adjacent sequence, e.g., heterologous polynucleotide (e.g.,modified nucleic acid encoding Factor IX). A promoter typicallyincreases an amount expressed from a heterologous polynucleotide ascompared to an amount expressed when no promoter exists.

An “enhancer” as used herein can refer to a sequence that is locatedadjacent to the heterologous polynucleotide. Enhancer elements aretypically located upstream of a promoter element but also function andcan be located downstream of or within a DNA sequence (e.g., aheterologous polynucleotide). Hence, an enhancer element can be located100 base pairs, 200 base pairs, or 300 or more base pairs upstream ordownstream of a heterologous polynucleotide. Enhancer elements typicallyincrease expressed of a heterologous polynucleotide above increasedexpression afforded by a promoter element.

Expression control elements (e.g., promoters) include those active in aparticular tissue or cell type, referred to herein as a “tissue-specificexpression control elements/promoters.” Tissue-specific expressioncontrol elements are typically active in specific cell or tissue (e.g.,liver, brain, central nervous system, spinal cord, eye, retina, bone,muscle, lung, pancreas, heart, kidney cell, etc.). Expression controlelements are typically active in these cells, tissues or organs becausethey are recognized by transcriptional activator proteins, or otherregulators of transcription, that are unique to a specific cell, tissueor organ type.

Examples of promoters active in skeletal muscle include promoters fromgenes encoding skeletal α-actin, myosin light chain 2A, dystrophin,muscle creatine kinase, as well as synthetic muscle promoters withactivities higher than naturally-occurring promoters (see, e.g., Li, etal., Nat. Biotech. 17:241-245 (1999)). Examples of promoters that aretissue-specific for liver are the human alpha 1-antitrypsin (hAAT)promoter; albumin, Miyatake, et al. J. Virol., 71:5124-32 (1997);hepatitis B virus core promoter, Sandig, et al., Gene Ther. 3:1002-9(1996); alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther.,7:1503-14 (1996)1, bone (osteocalcin, Stein, et al., Mol. Biol. Rep.,24:185-96 (1997); bone sialoprotein, Chen, et al., J. Bone Miner. Res.11:654-64 (1996)), lymphocytes (CD2, Hansal, et al., J. Immunol.,161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor a chain),neuronal (neuron-specific enolase (NSE) promoter, Andersen, et al.,Cell. Mol. Neurobiol., 13:503-15 (1993); neurofilament light-chain gene,Piccioli, et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991); theneuron-specific vgf gene, Piccioli, et al., Neuron, 15:373-84 (1995);among others. An example of an enhancer active in liver isapolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem.,272:29113-19 (1997)).

Expression control elements also include ubiquitous or promiscuouspromoters/enhancers which are capable of driving expression of apolynucleotide in many different cell types. Such elements include, butare not limited to the cytomegalovirus (CMV) immediate earlypromoter/enhancer sequences, the Rous sarcoma virus (RSV)promoter/enhancer sequences and the other viral promoters/enhancersactive in a variety of mammalian cell types, or synthetic elements thatare not present in nature (see, e.g., Boshart et al, Cell, 41:521-530(1985)), the SV40 promoter, the dihydrofolate reductase promoter, thecytoplasmic β-actin promoter and the phosphoglycerol kinase (PGK)promoter.

Expression control elements also can confer expression in a manner thatis regulatable, that is, a signal or stimuli increases or decreasesexpression of the operably linked heterologous polynucleotide. Aregulatable element that increases expression of the operably linkedpolynucleotide in response to a signal or stimuli is also referred to asan “inducible element” (i.e., is induced by a signal). Particularexamples include, but are not limited to, a hormone (e.g., steroid)inducible promoter. A regulatable element that decreases expression ofthe operably linked polynucleotide in response to a signal or stimuli isreferred to as a “repressible element” (i.e., the signal decreasesexpression such that when the signal, is removed or absent, expressionis increased). Typically, the amount of increase or decrease conferredby such elements is proportional to the amount of signal or stimulipresent; the greater the amount of signal or stimuli, the greater theincrease or decrease in expression. Particular non-limiting examplesinclude zinc-inducible sheep metallothionine (MT) promoter; the steroidhormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7polymerase promoter system (WO 98/10088); the tetracycline-repressiblesystem (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551(1992)); the tetracycline-inducible system (Gossen, et al., Science.268:1766-1769 (1995); see also Harvey, et al., Curr. Opin. Chem. Biol.2:512-518 (1998)); the RU486-inducible system (Wang, et al., Nat.Biotech. 15:239-243 (1997) and Wang, et al., Gene Ther. 4:432-441(1997)1; and the rapamycin-inducible system (Magari, et al., J. Clin.Invest. 100:2865-2872 (1997); Rivera, et al., Nat. Medicine. 2:1028-1032(1996)). Other regulatable control elements which may be useful in thiscontext are those which are regulated by a specific physiological state,e.g., temperature, acute phase, development.

Expression control elements also include the native elements(s) for theheterologous polynucleotide. A native control element (e.g., promoter)may be used when it is desired that expression of the heterologouspolynucleotide should mimic the native expression. The native elementmay be used when expression of the heterologous polynucleotide is to beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. Other nativeexpression control elements, such as introns, polyadenylation sites orKozak consensus sequences may also be used.

As used herein, the term “operable linkage” or “operably linked” refersto a physical or functional juxtaposition of the components so describedas to permit them to function in their intended manner. In the exampleof an expression control element in operable linkage with a nucleicacid, the relationship is such that the control element modulatesexpression of the nucleic acid. More specifically, for example, two DNAsequences operably linked means that the two DNAs are arranged (cis ortrans) in such a relationship that at least one of the DNA sequences isable to exert a physiological effect upon the other sequence.

Accordingly, modified nucleic acid sequences encoding human FIX protein,and vectors and plasmids, including viral vectors such as lenti- andparvovirus vectors, including AAV vectors, as well as compositionsthereof, can include additional nucleic acid elements. These elementsinclude, without limitation one or more copies of an AAV ITR sequence,an expression control (e.g., promoter/enhancer) element, a transcriptiontermination signal or stop codon, 5′ or 3′ untranslated regions (e.g.,polyadenylation (polyA) sequences) which flank a polynucleotidesequence, or an intron, such as all or a portion of intron I of genomichuman Factor IX (SEQ ID NO:13).

Nucleic acid elements further include, for example, filler or stufferpolynucleotide sequences, for example to improve packaging and reducethe presence of contaminating nucleic acid, e.g., to reduce packaging ofthe plasmid backbone. As disclosed herein, AAV vectors typically acceptinserts of DNA having a defined size range which is generally about 4 kbto about 5.2 kb, or slightly more. Thus, for shorter sequences,inclusion of a stuffer or filler in the insert fragment in order toadjust the length to near or at the normal size of the virus genomicsequence acceptable for AAV vector packaging into virus particle. Invarious embodiments, a filler/stuffer nucleic acid sequence is anuntranslated (non-protein encoding) segment of nucleic acid. Inparticular embodiments of an AAV vector, a heterologous polynucleotidesequence has a length less than 4.7 kb and the filler or stufferpolynucleotide sequence has a length that when combined (e.g., insertedinto a vector) with the heterologous polynucleotide sequence has a totallength between about 3.0-5.5 kb, or between about 4.0-5.0 Kb, or betweenabout 4.3-4.8 Kb.

An intron can also function as a filler or stuffer polynucleotidesequence in order to achieve a length for AAV vector packaging into avirus particle. Introns and intron fragments (e.g. portion of intron Iof FIX) that function as a filler or stuffer polynucleotide sequencealso can enhance expression. Inclusion of an intron element may enhanceexpression compared with expression in the absence of the intron element(Kurachi et al., 1995, supra).

The use of introns is not limited to the inclusion of Factor IX intron Isequences, but also include other introns, which introns may beassociated with the same gene (e.g., where the nucleic acid encodesFactor IX, the intron is derived from an intron present in Factor IXgenomic sequence) or associated with a completely different gene orother DNA sequence. Accordingly, other untranslated (non-proteinencoding) regions of nucleic acid, such as introns found in genomicsequences from cognate (related) genes (the heterologous polynucleotidesequence encodes all or a portion of same protein encoded by the genomicsequence) and non-cognate (unrelated) genes (the heterologouspolynucleotide sequence encodes a protein that is distinct from theprotein encoded by the genomic sequence) can also function as filler orstuffer polynucleotide sequences in accordance with the invention.

A “portion of intron I” as used herein, is meant region of intron Ihaving a nucleotide length of from about 0.1 kb to about 1.7 kb, whichregion enhances expression of Factor IX, typically by about 1.5-fold ormore on a plasmid or viral vector template when compared with expressionof FIX in the absence of a portion of intron I. A more specific portionis a 1.3 kb portion of intron I. A non-limiting example of a sequence ofFactor IX intron I is intron A, a chimera composed of the 5′ part andthe 3′ part of FIX first intron as set forth in SEQ ID NO:13.

Expression control elements, ITRs, poly A sequences, filler or stufferpolynucleotide sequences can vary in length. In particular aspects, anexpression control element, ITR, polyA, or a filler or stufferpolynucleotide sequence is a sequence between about 1-10, 10-20, 20-30,30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300,300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, or2,000-2,500 nucleotides in length.

According to one non-limiting embodiment, an AAV vector comprises an AAVcapsid comprising the 4-1 capsid variant VP1 protein (SEQ ID NO:4) and agenome for expressing a heterologous gene in a transduced mammaliancell.

The capsid of this vector may further comprise the VP2 and VP3 proteinsfrom the 4-1 capsid variant (SEQ ID NO:27 and SEQ ID NO:3,respectively). According to certain non-limiting embodiments, the VP1protein and VP2 proteins are in a stoichiometric ratio of approximately1:1 (or some other ratio), and the VP3 protein is in a ratio to eitherVP1 or VP2, or both VP1 and VP2, in an approximate ratio of about 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, or some other ratio.

In some embodiments, a genome of an AAV vector, including withoutlimitation one having a capsid comprising the 4-1 capsid variantproteins (VP1, VP2, VP3), comprises a heterologous nucleic acid sequenceencoding human Factor IX (FIX) protein. In some embodiments, FIX proteinis wild type, and in other embodiments FIX protein contains asubstitution mutation or other mutation that alters the protein'sactivity. In some embodiments, the mutation increases FIX catalyticactivity and/or activity of the protein as a procoagulant. In someembodiments, FIX protein is the Padua FIX protein, with an Arg to Alasubstitution at the amino acid corresponding to position 338 of FIXprotein. According to some embodiments, a gene encoding human FIX(including FIX Padua) is codon optimized, for example, by reducing oreven eliminating CpG dinucleotides. Other types of codon optimizationare possible as well.

In some embodiments, a genome of the AAV vector further comprisesinverted terminal repeats (ITRs) from AAV2 positioned at the left andright ends (i.e., 5′ and 3′ termini, respectively) of the genome. Insome embodiments, a left ITR comprises or consists of nucleotides 1-141from SEQ ID NO:12 (disclosed herein as SEQ ID NO:13), and a right ITRcomprises or consists of nucleotides 4097-4204 from SEQ ID NO:12(disclosed herein as SEQ ID NO:20). Each ITR may be separated from otherelements in the vector genome by a nucleic acid sequence of variablelength.

In other embodiments, a genome of the AAV vector further comprises anexpression control element, including a promoter, and optionally anenhancer. In some embodiments, an AAV vector genome comprises both apromoter and an enhancer, which may be constituitive, inducible, ortissue specific. In some embodiments, a promoter, or an enhancer, orboth are tissue specific. According to an exemplary embodiment, bothenhancer and promoter are preferentially active in hepatocytes, comparedto certain other cell types. According to one embodiment, an enhancer isall or a portion of the human ApoE HCR-1 enhancer, and a promoter is allor a portion of the human alpha-1 antitrypsin (AAT) promoter. In someembodiments, an AAV vector genome includes a human ApoE HCR-1 enhancercomprising or consisting of nucleotides 152-472 from SEQ ID NO:12(disclosed herein as SEQ ID NO:14), and includes a human AAT promotercomprising or consisting of nucleotides 482-878 from SEQ ID NO:12(disclosed herein as SEQ ID NO:15). In some embodiments, an ApoE HCR-1enhancer is positioned 5′ of an AAT promoter, and the sequences may becontiguous, or separated by another nucleotide sequence. According tosome embodiments, an enhancer and promoter are positioned 5′ of anucleic acid sequence coding Factor IX, and may be contiguously joinedto the first exon of a Factor IX gene, or may be separated therefrom by5′ untranslated sequence (UTR) from a human Factor IX gene, or someother sequence serving as a spacer. In an exemplary non-limitingembodiment, a 5′ UTR sequence comprises or consists of nucleotides879-907 from SEQ ID NO:12.

In some embodiments, gene encoding FIX, including naturally occurringFIX Padua, includes one or more introns present in a human Factor IXgenomic sequence. In other embodiments, all introns may be excluded, anexample of which is disclosed as the nucleic acid sequence of SEQ IDNO:10 and referred to herein as the coding sequence for “FIX39.” Ifpresent, an intron can behave as a stuffer or filler sequence asdescribed herein. The entire gene can be codon-optimized to deplete oreliminate CpG dinucleotides.

In a particular non-limiting embodiment, a gene encoding human Factor IXused in an AAV vector comprises or consists of nucleotides 908-3731 fromSEQ ID NO:12, which encodes the FIX Padua and is codon-optimized toeliminate CpG dinucleotides. This sequence includes an exon 1(nucleotides 908-995 from SEQ ID NO:12), a first intron (sometimes knownas intron I; nucleotides 996-2433 from SEQ ID NO:12), and exons 2-8(nucleotides 2434-3731 from SEQ ID NO:12).

In certain embodiments, a gene encoding Factor IX may be followed at its3′ end by 3′ UTR sequence from a human Factor IX gene (such as withoutlimitation nucleotides 3732-3779 from SEQ ID NO:12 and/or by apolyadenylate (polyA) sequence from a Factor IX gene, or another gene.In one non-limiting example, the polyA sequence can be from the bovinegrowth hormone (bGH) gene, and can comprise or consist of nucleotides3820-4047 from SEQ ID NO:12. In some embodiments, a 3′UTR can bevariably spaced from the polyA sequence by an intervening sequence ofnucleotides.

In some embodiments, the elements described above can be combined intoone AAV vector genome. According to one non-limiting example, an AAVvector can have a genome comprising, in 5′ to 3′ order, a left AAV ITR,the ApoE HCR-1 enhancer (or portion thereof), the hAAT promoter (orportion thereof), a portion of human Factor IX 5′UTR, nucleic acidencoding human Factor IX Padua (including optionally one or moreintrons, such as intron I), a portion of human Factor IX 3′ UTR, a polyAsequence from bGH (or portion thereof), and at the right an AAV2 ITR. Insome of these embodiments, a left AAV2 ITR has the nucleic acid sequenceof SEQ ID NO:13; an ApoE HCR-1 enhancer has the nucleic acid sequence ofSEQ ID NO:14; a hAAT promoter has the nucleic acid sequence of SEQ IDNO:15; a 5′ UTR has the nucleic acid sequence of SEQ ID NO:16; a geneencoding FIX Padua (including intron I) encodes the FIX protein encodedby nucleic acid sequence of SEQ ID NO:10; the 3′ UTR has a nucleic acidsequence of SEQ ID NO:18; a polyA region has the nucleic acid sequenceof SEQ ID NO:19; and a right AAV2 ITR has a nucleic acid sequence of SEQID NO:20.

According to certain embodiments, a genome of an AAV vector comprises orconsists of nucleotides 1-4204 from SEQ ID NO:12, or a sequence that isat least 95%, 96%, 97%, 98%, or 99% identical thereto. In some of theseembodiments, a capsid comprises the 4-1 VP1 capsid protein variant (SEQID NO:4) and the corresponding VP2 and VP2 capsid proteins. In aparticular non-limiting embodiment of an AAV vector, referred to hereinas “AAV-FIX39-Padua,” the vector includes a capsid formed from 4-1capsid variant proteins (VP1, VP2, VP3), and a single-stranded genomecomprising a nucleic acid sequence corresponding to nucleotides 1-4204from SEQ ID NO:12.

AAV “empty capsids” as used herein do not contain a vector genome(hence, the term “empty”), in contrast to “genome containing capsids”which contain an AAV vector genome. Empty capsids are virus-likeparticles in that they react with one or more antibodies that reactswith the intact (genome containing AAV vector) virus.

Empty capsids can be included in AAV vector preparations. If desired,AAV empty capsids can be added to AAV vector preparations, oradministered separately to a subject in accordance with the methodsherein.

Although not wishing to be bound by theory, AAV empty capsids arebelieved to bind to or react with antibodies against the AAV vectors,thereby functioning as a decoy to reduce inactivation of the AAV vector.Such a decoy acts to absorb antibodies directed against the AAV vectorthereby increasing or improving AAV vector transgene transduction ofcells (introduction of the transgene), and in turn increased cellularexpression of the transcript and/or encoded protein.

Empty capsids can be generated and purified at a quality and theirquantities determined. For example, empty capsid titer can be measuredby spectrophotometry by optical density at 280 nm wavelength (based onSommer et al., Mol. Ther. 2003 January; 7(1):122-8).

Empty-AAV or empty capsids are sometimes naturally found in AAV vectorpreparations. Such natural mixtures can be used in accordance with theinvention, or if desired be manipulated to increase or decrease theamount of empty capsid and/or vector. For example, the amount of emptycapsid can optionally be adjusted to an amount that would be expected toreduce the inhibitory effect of antibodies that react with an AAV vectorthat is intended to be used for vector-mediated gene transduction in thesubject. The use of empty capsids is described in US Publication2014/0336245.

In various embodiments, AAV empty capsids are formulated with AAVvectors and/or administered to a subject. In particular aspects, AAVempty capsids are formulated with less than or an equal amount of vector(e.g., .about 1.5 to 100-fold AAV vectors to AAV empty capsids, or abouta 1:1 ratio of AAV vectors to AAV empty capsids). In other particularaspects, AAV vectors are formulated with an excess of AAV empty capsids(e.g., greater than 1 fold AAV empty capsids to AAV vectors, e.g., 1.5to 100-fold AAV empty capsids to AAV vectors). Optionally, subjects withlow to negative titer AAV NAb can receive lower amounts of empty capsids(1 to 10 fold AAV empty capsids to AAV vectors, 2-6 fold AAV emptycapsids to AAV vectors, or about 4-5 fold AAV empty capsids to AAVvectors).

According to certain embodiments, pharmaceutical compositions comprisingAAV vectors include those comprising the 4-1 capsid variant proteins(VP1, VP2 and VP3), comprise an excess of empty capsids greater than theconcentration of AAV vectors (i.e., those containing a vector genome) inthe composition. A ratio of empty capsids to AAV vectors can be about1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10 to 1, or some other ratio.

In some embodiments, empty capsids comprise the same VP1, VP2, and VP3capsid proteins that are present in the AAV vectors. In otherembodiments, empty capsids comprise VP1, VP2 and VP3 proteins having adifferent amino acid sequence than those found in the AAV vectors.Typically, although not necessarily, if the capsid proteins of the emptycapsids and capsids of the AAV vectors are not identical in sequence,they will be of the same serotype.

According to some embodiments, a composition comprises an AAV vectordescribed herein as AAV-FIX39-Padua (or one having the same capsid and agenome sequence at least 95%, 96%, 97%, 98% or 99% identical thereto)and optionally an excess of empty capsids comprising the same capsidproteins, wherein the ratio of empty capsids to the AAV vector is about1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10 to 1, or some other ratio. In someembodiments, the ratio in the composition of AAV-FIX39-Padua to emptycapsids is about 1:5. In other embodiments, compositions comprisingAAV-FIX39-Padua and empty capsids are administered to a human subjecthaving hemophilia B, including severe, moderate, or mild hemophilia B.

A “selectable marker gene” refers to a gene that when expressed confersa selectable phenotype, such as antibiotic resistance (e.g., kanamycin),on a transduced cell. A “reporter” gene is one that provides adetectable signal. A non-limiting example of a reporter gene is theluciferase gene.

Nucleic acid, polynucleotides, expression vectors (e.g., vectorgenomes), plasmids, including modified forms can be made using variousstandard cloning, recombinant DNA technology, via cell expression or invitro translation and chemical synthesis techniques. Purity ofpolynucleotides can be determined through sequencing, gelelectrophoresis and the like. For example, nucleic acids can be isolatedusing hybridization or computer-based database screening techniques.Such techniques include, but are not limited to: (1) hybridization ofgenomic DNA or cDNA libraries with probes to detect homologousnucleotide sequences; (2) antibody screening to detect polypeptideshaving shared structural features, for example, using an expressionlibrary; (3) polymerase chain reaction (PCR) on genomic DNA or cDNAusing primers capable of annealing to a nucleic acid sequence ofinterest; (4) computer searches of sequence databases for relatedsequences; and (5) differential screening of a subtracted nucleic acidlibrary.

The term “isolated,” when used as a modifier of a composition, meansthat the compositions are made by the hand of man or are separated,completely or at least in part, from their naturally occurring in vivoenvironment. Generally, isolated compositions are substantially free ofone or more materials with which they normally associate with in nature,for example, one or more protein, nucleic acid, lipid, carbohydrate,cell membrane. The term “isolated” does not exclude combinationsproduced by the hand of man, for example, a recombinant vector (e.g.,rAAV) sequence, or virus particle that packages or encapsidates a vectorgenome and a pharmaceutical formulation. The term “isolated” also doesnot exclude alternative physical forms of the composition, such ashybrids/chimeras, multimers/oligomers, modifications (e.g.,phosphorylation, glycosylation, lipidation) or derivatized forms, orforms expressed in host cells produced by the hand of man.

Methods and uses of the invention provide a means for delivering(transducing) heterologous polynucleotides (transgenes) into host cells,including dividing and/or non-dividing cells. The recombinant vector(e.g., rAAV) sequences, vector genomes, recombinant virus particles,methods, uses and pharmaceutical formulations of the invention areadditionally useful in a method of delivering, administering orproviding a nucleic acid, or protein to a subject in need thereof, as amethod of treatment. In this manner, the nucleic acid is transcribed andthe protein may be produced in vivo in a subject. The subject maybenefit from or be in need of the nucleic acid or protein because thesubject has a deficiency of the nucleic acid or protein, or becauseproduction of the nucleic acid or protein in the subject may impart sometherapeutic effect, as a method of treatment or otherwise.

In general, recombinant lenti- or parvo-virus vector (e.g., AAV)sequences, vector genomes, recombinant virus particles, methods and usesmay be used to deliver any heterologous polynucleotide (transgene) witha biological effect to treat or ameliorate one or more symptomsassociated with any disorder related to insufficient or undesirable geneexpression. Recombinant lenti- or parvo-virus vector (e.g., AAV)sequences, plasmids, vector genomes, recombinant virus particles,methods and uses may be used to provide therapy for various diseasestates.

Invention nucleic acids, vectors, recombinant vectors (e.g., rAAV),vector genomes, and recombinant virus particles, methods and uses permitthe treatment of genetic diseases. In general, disease states fall intotwo classes: deficiency states, usually of enzymes, which are generallyinherited in a recessive manner, and unbalanced states, at leastsometimes involving regulatory or structural proteins, which areinherited in a dominant manner. For deficiency state diseases, genetransfer could be used to bring a normal gene into affected tissues forreplacement therapy, as well as to create animal models for the diseaseusing antisense mutations. For unbalanced disease states, gene transfercould be used to create a disease state in a model system, which couldthen be used in efforts to counteract the disease state. The use ofsite-specific integration of nucleic acid sequences to correct defectsis also possible.

Illustrative examples of disease states include, but are not limited to:blood coagulation disorders such as hemophilia A, hemophilia B,thalassemia, and anemia.

In accordance with the invention, treatment methods and uses areprovided that include invention nucleic acids, vectors, recombinantvectors (e.g., rAAV), vector genomes, and recombinant virus particles.Methods and uses of the invention are broadly applicable to providing,or increasing or stimulating, gene expression or function, e.g., geneaddition or replacement.

In one embodiment, a method or use of the invention includes: (a)providing a modified nucleic acid encoding Factor IX, such as FIX with areduced number of CpG dinucleotides, such as in a vector or a vectorgenome, wherein the modified nucleic acid sequence is operably linked toan expression control element conferring transcription of said sequence;and (b) administering an amount of the modified nucleic acid to themammal such that Factor IX is expressed in the mammal.

In another embodiment, a method or use of the invention includesdelivering or transferring a modified nucleic acid encoding Factor IXsequence, such as FIX with a reduced number of CpG dinucleotides, into amammal or a cell of a mammal, by administering a viral (e.g., AAV)particle or plurality of viral (e.g., AAV) particles (e.g., such ascapsid variants (e.g., 4-1)) comprising a vector genome, the vectorgenome comprising the modified nucleic acid encoding Factor IX, such asFIX with a reduced number of CpG dinucleotides (and optionally an ITR,intron, polyA, a filler/stuffer polynucleotide sequence) to a mammal ora cell of a mammal, thereby delivering or transferring the modifiednucleic acid encoding Factor IX into the mammal or cell of the mammal.

In particular aspects of invention methods and uses disclosed herein,expression of the nucleic acid provides a therapeutic benefit to themammal (e.g., human). In a more particular aspect, expression of FactorIX provides a therapeutic benefit to the mammal (e.g., human), such as amammal that has hemophilia B. In various further particular aspects, afiller/stuffer polynucleotide sequence is included in the vectorsequence such that the combined length with the modified nucleic acidencoding Factor IX, such as FIX with a reduced number of CpGdinucleotides, has a total length of between about 3.0 Kb-5.5 Kb, orbetween about 4.0 Kb-5.0 Kb, or between about 4.3 Kb-4.8 kb.

Methods and uses of the invention include treatment methods, whichresult in any therapeutic or beneficial effect. In various inventionmethods and uses, further included are inhibiting, decreasing orreducing one or more adverse (e.g., physical) symptoms, disorders,illnesses, diseases or complications caused by or associated with thedisease. For a bleeding disorder such as hemophilia, a therapeutic orbeneficial effect includes, but is not limited to, reduced bruising,reduced blood clotting time, reduced bleeding episodes (duration,severity, frequency). For example, reduced duration, severity orfrequency of joint or cerebral (brain) bleeding episodes. For a bleedingdisorder such as hemophilia, a therapeutic or beneficial effect alsoincludes, but is not limited to, reduced dosage of a supplementalclotting factor protein (e.g., Factor IX protein) or elimination ofadministration of a supplemental clotting factor protein (e.g., FactorIX protein).

A therapeutic or beneficial effect of treatment is therefore anyobjective or subjective measurable or detectable improvement or benefitprovided to a particular subject. A therapeutic or beneficial effect canbut need not be complete ablation of all or any particular adversesymptom, disorder, illness, or complication of a disease. Thus, asatisfactory clinical endpoint is achieved when there is an incrementalimprovement or a partial reduction in an adverse symptom, disorder,illness, or complication caused by or associated with a disease, or aninhibition, decrease, reduction, suppression, prevention, limit orcontrol of worsening or progression of one or more adverse symptoms,disorders, illnesses, or complications caused by or associated with thedisease, over a short or long duration (hours, days, weeks, months,etc.).

Compositions, such as nucleic acids, vectors, recombinant vectors (e.g.,rAAV), vector genomes, and recombinant virus particles including vectorgenomes, and methods and uses of the invention, can be administered in asufficient or effective amount to a subject in need thereof. An“effective amount” or “sufficient amount” refers to an amount thatprovides, in single or multiple doses, alone or in combination, with oneor more other compositions (therapeutic agents such as a drug),treatments, protocols, or therapeutic regimens agents, a detectableresponse of any duration of time (long or short term), an expected ordesired outcome in or a benefit to a subject of any measurable ordetectable degree or for any duration of time (e.g., for minutes, hours,days, months, years, or cured).

One skilled in the art can determine whether administration of a singlerAAV/vector dose is sufficient or whether are to administer multipledoses of rAAV/vector. For example, if FIX levels decreases below apre-determined level (e.g., less than the minimum that provides atherapeutic benefit), one skilled in the art can determine ifappropriate to administer additional doses of rAAV/vector.

The dose to achieve a therapeutic effect, e.g., the dose in vectorgenomes/per kilogram of body weight (vg/kg), will vary based on severalfactors including, but not limited to: route of administration, thelevel of heterologous polynucleotide expression required to achieve atherapeutic effect, the specific disease treated, any host immuneresponse to the viral vector, a host immune response to the heterologouspolynucleotide or expression product (protein), and the stability of theprotein expressed. One skilled in the art can determine a rAAV/vectorgenome dose range to treat a patient having a particular disease ordisorder based on the aforementioned factors, as well as other factors.Generally, doses will range from at least 1×10⁸, or more, for example,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³ or 1×10¹⁴, or more, vector genomesper kilogram (vg/kg) of the weight of the subject, to achieve atherapeutic effect.

In some embodiments, a therapeutically effective dose of an AAV vector,including, for example, AAV-FIX39-Padua, or one having the same capsidand a genome sequence at least 95%, 96%, 97%, 98% or 99% identicalthereto, is one that is sufficient, when administered to a subject, forexample, a human, with hemophilia B or other deficiency of Factor IXactivity, to convert severe hemophilia B to moderate or mild hemophiliaB, or even to result in an apparently disease-free state. In otherembodiments, a therapeutically effective dose of an AAV vector is onethat is sufficient to allow a human subject with hemophilia B to foregoFactor IX replacement therapy entirely, or reduce the frequency withwhich replacement FIX is administered to maintain adequate hemostasis.As understood by those of skill in the art, factor replacement therapyis the current standard of care for hemophilia B, but requires frequentinjections of recombinantly produced human Factor IX to compensate forthe patient's inability to produce sufficient levels of functionalclotting factor.

It is generally accepted that severe hemophilia B is characterized byfrequent bleeding (for example, at least once or twice per week), oftenspontaneously (without preceding trauma), into a subject's muscles orjoints. Less than 1% of FIX activity found in healthy humans isassociated with severe hemophilia B. It is generally accepted that humansubjects with moderate hemophilia B bleed less frequently than thosewith severe hemophilia B, for example, about once per month, but willbleed for a longer time than normal after surgery, trauma, or dentalwork. It is generally accepted that human subjects with moderate diseasedo not often bleed spontaneously. FIX activity 1%-5% of normal isassociated with moderate hemophilia B. Generally, human subjects withmild hemophilia B bleed excessively, if at all, only as a result ofsurgery or major trauma. Generally, mild hemophilia is associated with6%-40% of normal FIX activity. Generally, individuals who are consideredhealthy, having no symptoms of hemophilia B, have a range of about 50%to 150% of normal FIX activity. Additional information can be found inFijnvandraat, et al., Diagnosis and management of hemophilia, Br. Med.J., 344:36-40 (2012).

Factor IX activity can be measured in a variety of ways known to thoseof skill in the art. For example, one exemplary non-limiting assay isthe one-stage activated partial thromboplastin time (APTT) assay todetermine FIX clotting activity in a plasma sample obtained from asubject. FIX activity is frequently expressed in international units(IU), where 1 IU is defined as the FIX clotting activity present in 1 mlof pooled plasma from normal donors. Using this convention, severehemophilia B is associated with less than 0.01 IU/ml FIX levels,moderate disease is associated with 0.02-0.05 IU/ml FIX levels, milddisease is associated with 0.06-0.40 IU/ml FIX levels, and beingdisease-free is associated with 0.50-1.50 IU/ml FIX levels.

As will be appreciated by one of skill in the art, a Factor IX variant,such as naturally occurring FIX Padua variant, that has higher catalyticactivity compared to wild type human FIX, can produce a given level ofFIX activity (e.g., 1 IU/ml) at a lower concentration of active proteincompared to “non-Padua” FIX.

In certain embodiments, a therapeutically effective dose of an AAVvector, including, for example, AAV-FIX39-Padua, or one having the samecapsid and a genome sequence at least 95%, 96%, 97%, 98% or 99%identical thereto, is one that is sufficient, when administered to asubject, for example, a human, with severe, moderate or mild hemophiliaB, to achieve plasma FIX activity that is about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or50%, or more of normal FIX activity. In other embodiments, atherapeutically effective dose is one that achieves 1% or greater FIXactivity in a subject otherwise lacking such activity, for example, from1.5-10%, 10-15%, 15-20%, 20-25%, 25-30% or greater FIX activity in asubject.

With respect to treating a subject with hemophilia B, a therapeuticallyeffective dose of an AAV vector including, for example, AAV-FIX39-Padua,or one having the same capsid and a genome sequence at least 98% or 99%identical thereto, may be at least 1×10¹⁰ vector genomes (vg) perkilogram (vg/kg) of the weight of the subject, or between about 1×10¹⁰to 1×10¹¹ vg/kg of the weight of the subject, or between about 1×10¹¹ to1×10¹² vg/kg (e.g., about 1×10¹¹ to 2×10¹¹ vg/kg or about 2×10¹¹ to3×10¹¹ vg/kg or about 3×10¹¹ to 4×10¹¹ vg/kg or about 4×10¹¹ to 5×10¹¹vg/kg or about 5×10¹¹ to 6×10¹¹ vg/kg or about 6×10¹¹ to 7×10¹¹ vg/kg orabout 7×10¹¹ to 8×10¹¹ vg/kg or about 8×10¹¹ to 9×10¹¹ vg/kg or about9×10¹¹ to 1×10¹² vg/kg) of the weight of the subject, or between about1×10¹² to 1×10¹³ vg/kg of the weight of the subject, to achieve adesired therapeutic effect. Additional doses can be in a range of about5×10¹⁰ to 1×10¹⁰ vector genomes (vg) per kilogram (vg/kg) of the weightof the subject, or in a range of about 1×10¹⁰ to 5×10¹¹ vg/kg of theweight of the subject, or in a range of about 5×10¹¹ to 1×10¹² vg/kg ofthe weight of the subject, or in a range of about 1×10¹² to 5×10¹³ vg/kgof the weight of the subject, to achieve a desired therapeutic effect.In other embodiments, a therapeutically effective dose of an AAV vectoris about 2.0×10¹¹ vg/kg, 2.1×10¹¹ vg/kg, 2.2×10¹¹ vg/kg, 2.3×10¹¹ vg/kg,2.4×10¹¹ vg/kg, 2.5×10¹¹ vg/kg, 2.6×10¹¹ vg/kg, 2.7×10¹¹ vg/kg, 2.8×10¹¹vg/kg, 2.9×10¹¹ vg/kg, 3.0×10¹¹ vg/kg, 3.1×10¹¹ vg/kg, 3.2×10¹¹ vg/kg,3.3×10¹¹ vg/kg, 3.4×10¹¹ vg/kg, 3.5×10¹¹ vg/kg, 3.6×10¹¹ vg/kg, 3.7×10¹¹vg/kg, 3.8×10¹¹ vg/kg, 3.9×10¹¹ vg/kg, 4.0×10¹¹ vg/kg, 4.1×10¹¹ vg/kg,4.2×10¹¹ vg/kg, 4.3×10¹¹ vg/kg, 4.4×10¹¹ vg/kg, 4.5×10¹¹ vg/kg, 4.6×10¹¹vg/kg, 4.7×10¹¹ vg/kg, 4.8×10¹¹ vg/kg, 4.9×10¹¹ vg/kg, 5.0×10¹¹ vg/kg,5.1×10¹¹ vg/kg, 5.2×10¹¹ vg/kg, 5.3×10¹¹ vg/kg, 5.4×10¹¹ vg/kg, 5.5×10¹¹vg/kg, 5.6×10¹¹ vg/kg, 5.7×10¹¹ vg/kg, 5.8×10¹¹ vg/kg, 5.9×10¹¹ vg/kg,6.0×10¹¹ vg/kg, 6.1×10¹¹ vg/kg, 6.2×10¹¹ vg/kg, 6.3×10¹¹ vg/kg, 6.4×10¹¹vg/kg, 6.5×10¹¹ vg/kg, 6.6×10¹¹ vg/kg, 6.7×10¹¹ vg/kg, 6.8×10¹¹ vg/kg,6.9×10¹¹ vg/kg, 7.0×10¹¹ vg/kg, 7.1×10¹¹ vg/kg, 7.2×10¹¹ vg/kg, 7.3×10¹¹vg/kg, 7.4×10¹¹ vg/kg, 7.5×10¹¹ vg/kg, 7.6×10¹¹ vg/kg, 7.7×10¹¹ vg/kg,7.8×10¹¹ vg/kg, 7.9×10¹¹ vg/kg, or 8.0×10¹¹ vg/kg, or some other dose.In any of these embodiments, an AAV vector can be AAV-FIX39-Padua, or anAAV vector having the same capsid and a genome sequence at least 95%,96%, 97%, 98% or 99% identical thereto, which may be administered to asubject in a pharmaceutically acceptable composition alone, or withempty capsids of the same capsid type at an empty to vector ratio ofabout 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some other ratio.

The doses of an “effective amount” or “sufficient amount” for treatment(e.g., to ameliorate or to provide a therapeutic benefit or improvement)typically are effective to provide a response to one, multiple or alladverse symptoms, consequences or complications of the disease, one ormore adverse symptoms, disorders, illnesses, pathologies, orcomplications, for example, caused by or associated with the disease, toa measurable extent, although decreasing, reducing, inhibiting,suppressing, limiting or controlling progression or worsening of thedisease is a satisfactory outcome.

An effective amount or a sufficient amount can but need not be providedin a single administration, may require multiple administrations, and,can but need not be, administered alone or in combination with anothercomposition (e.g., agent), treatment, protocol or therapeutic regimen.For example, the amount may be proportionally increased as indicated bythe need of the subject, type, status and severity of the diseasetreated or side effects (if any) of treatment. In addition, an effectiveamount or a sufficient amount need not be effective or sufficient ifgiven in single or multiple doses without a second composition (e.g.,another drug or agent), treatment, protocol or therapeutic regimen,since additional doses, amounts or duration above and beyond such doses,or additional compositions (e.g., drugs or agents), treatments,protocols or therapeutic regimens may be included in order to beconsidered effective or sufficient in a given subject. Amountsconsidered effective also include amounts that result in a reduction ofthe use of another treatment, therapeutic regimen or protocol, such asadministration of recombinant clotting factor protein for treatment of aclotting disorder (e.g., hemophilia A or B).

An effective amount or a sufficient amount need not be effective in eachand every subject treated, nor a majority of treated subjects in a givengroup or population. An effective amount or a sufficient amount meanseffectiveness or sufficiency in a particular subject, not a group or thegeneral population. As is typical for such methods, some subjects willexhibit a greater response, or less or no response to a given treatmentmethod or use.

The term “ameliorate” means a detectable or measurable improvement in asubject's disease or symptom thereof, or an underlying cellularresponse. A detectable or measurable improvement includes a subjectiveor objective decrease, reduction, inhibition, suppression, limit orcontrol in the occurrence, frequency, severity, progression, or durationof the disease, or complication caused by or associated with thedisease, or an improvement in a symptom or an underlying cause or aconsequence of the disease, or a reversal of the disease.

Thus, a successful treatment outcome can lead to a “therapeutic effect,”or “benefit” of decreasing, reducing, inhibiting, suppressing, limiting,controlling or preventing the occurrence, frequency, severity,progression, or duration of a disease, or one or more adverse symptomsor underlying causes or consequences of the disease in a subject.Treatment methods and uses affecting one or more underlying causes ofthe disease or adverse symptoms are therefore considered to bebeneficial. A decrease or reduction in worsening, such as stabilizingthe disease, or an adverse symptom thereof, is also a successfultreatment outcome.

A therapeutic benefit or improvement therefore need not be completeablation of the disease, or any one, most or all adverse symptoms,complications, consequences or underlying causes associated with thedisease. Thus, a satisfactory endpoint is achieved when there is anincremental improvement in a subject's disease, or a partial decrease,reduction, inhibition, suppression, limit, control or prevention in theoccurrence, frequency, severity, progression, or duration, or inhibitionor reversal of the disease (e.g., stabilizing one or more symptoms orcomplications), over a short or long duration of time (hours, days,weeks, months, etc.). Effectiveness of a method or use, such as atreatment that provides a potential therapeutic benefit or improvementof a disease, can be ascertained by various methods, such as blood clotformation time, etc.

According to some embodiments, a therapeutically effective dose of anAAV vector is one that is sufficient, when administered to a humansubject with hemophilia B, to result in FIX activity above a certainlevel for a sustained period of time. In some of these embodiments, aneffective dose of an AAV vector results in at least 1% normal FIXactivity in human subjects with hemophilia B for a sustained period ofat least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 1.5 years, 2 years, 2.5 years, 3 years,3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, ormore. In other embodiments, an effective dose of an AAV vector resultsin at least 5% normal FIX activity for a sustained period of at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10years, or more. In other embodiments, an effective dose of an AAV vectorresults in at least 10% normal FIX activity for a sustained period of atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, orat least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10 years, or more. In other embodiments, an effective dose of anAAV vector results in at least 15% normal FIX activity for a sustainedperiod of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10 years, or more. In other embodiments, aneffective dose of an AAV vector results in at least 20% normal FIXactivity for a sustained period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or more. In otherembodiments, an effective dose of an AAV vector results in at least 25%normal FIX activity for a sustained period of at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or more.In other embodiments, an effective dose of an AAV vector results in atleast 30% normal FIX activity for a sustained period of at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, or at least1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10years, or more. In other embodiments, an effective dose of an AAV vectorresults in at least 35% normal FIX activity for a sustained period of atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 months, orat least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10 years, or more. In other embodiments, an effective dose of anAAV vector results in at least 40% normal FIX activity for a sustainedperiod of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10 years, or more. In other embodiments, aneffective dose of an AAV vector results in at least 45% normal FIXactivity for a sustained period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, or 17 months, or at least 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 years, or more. In anyof these embodiments, the AAV vector can be AAV-FIX39-Padua, or an AAVvector having the same capsid and a genome sequence at least 95%, 96%,97%, 98% or 99% identical thereto, which may be administered to asubject in a pharmaceutically acceptable composition alone, or withempty capsids of the same capsid type at an empty to vector ratio ofabout 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some other ratio.

According to other embodiments, a therapeutically effective dose of anAAV vector is one that is sufficient, when administered to a humansubject with severe or moderate hemophilia B, to result in FIX activitythat is at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or45%, of normal for a sustained period of at least 6 months. In someembodiments, the dose of AAV-FIX39-Padua, or an AAV vector having thesame capsid and a genome sequence at least 95%, 96%, 97%, 98% or 99%identical thereto, is about 5.0×10¹¹ vg/kg, which may be administered ina pharmaceutically acceptable composition alone, or with empty capsidsof the same capsid type at an empty to vector ratio of about 2:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some other ratio.

It may be seen that in some human subjects that have received atherapeutically effective dose of an AAV vector, including for example,AAV-FIX39-Padua, or an AAV vector having the same capsid and a genomesequence at least 95%, 96%, 97%, 98% or 99% identical thereto, that FIXactivity attributable to the vector declines over an extended period oftime (e.g., months or years) to a level that is no longer deemedsufficient (for example, where the subject exhibits symptoms and/or FIXactivity characteristic of moderate or severe hemophilia B). In suchcircumstances, the subject can be dosed again with the same type of AAVvector as in the initial treatment. In other embodiments, particularlyif the subject has developed an immune reaction to the initial vector,the patient may be dosed with an AAV vector designed to express FIX intarget cells, but having a capsid of a different or variant serotypethat is less immunoreactive compared to the first AAV vector.

According to certain embodiments, a therapeutically effective dose of anAAV vector is one that is sufficient, when administered to a humansubject with hemophilia B, to reduce or even eliminate the subject'sneed for recombinant human Factor IX replacement therapy to maintainadequate hemostasis. Thus, in some embodiments, a therapeuticallyeffective dose of an AAV vector can reduce the frequency with which anaverage human subject having moderate or severe hemophilia B needs FIXreplacement therapy to maintain adequate hemostasis by about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%. In related embodiments, a therapeuticallyeffective dose of an AAV vector can reduce the dose of recombinant humanFactor IX that an average human subject having moderate or severehemophilia B needs to maintain adequate hemostasis by about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%. In any of these embodiments, the AAV vector canbe AAV-FIX39-Padua, or an AAV vector having the same capsid and a genomesequence at least 95%, 96%, 97%, 98% or 99% identical thereto, which maybe administered to a subject in a pharmaceutically acceptablecomposition alone, or with empty capsids of the same capsid type at anempty to vector ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, or some other ratio.

In other embodiments, a therapeutically effective dose of an AAV vectoris one that is sufficient, when administered to a human subject withsevere hemophilia B, to reduce or even eliminate spontaneous bleedinginto the joints. Thus, in some embodiments, a therapeutically effectivedose of an AAV vector can reduce the frequency of spontaneous bleedinginto the joints of a human subject with severe hemophilia B by about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100%, compared to the average untreated humansubject with severe hemophilia B. Bleeding into the joints can bedetected using magnetic resonance imaging or ultrasonography of thejoints, or other techniques familiar to those of skill in the art. Inany of these embodiments, the AAV vector can be AAV-FIX39-Padua, or anAAV vector having the same capsid and a genome sequence at least 95%,96%, 97%, 98% or 99% identical thereto, which may be administered to asubject in a pharmaceutically acceptable composition alone, or withempty capsids of the same capsid type at an empty to vector ratio ofabout 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some other ratio.

Prior efforts to develop AAV vectors to treat hemophilia have beenunsuccessful, at least in part it is believed, because of a robustimmune response to AAV capsid in prior clinical trials. (See, forexample, Nathwani, et al., NEJM 2011; 365(25):2357-2365; and Manno, etal., Nat Med 2006; 12(3):342-347). A clinical trial underway hasdemonstrated that certain embodiments of AAV vectors herein can producea high level of FIX activity in human subjects with severe hemophilia B,while resulting in no or minimal immune response even as much as 6months after the AAV vectors were administered (Example 5). Thus,according to certain embodiments, a therapeutically effective dose of anAAV vector is one that when administered to a subject with severe ormoderate hemophilia B results in FIX activity adequate to maintainhemostasis, while producing no or minimal immune response over asignificant period of time. In certain embodiments, the immune responsecan be an innate immune response, a humoral immune response, or acellular immune response, or even all three types of immune response. Insome embodiments, the immune response can be against the capsid, vectorgenome, and/or Factor IX protein produced from transduced cells.

According to certain embodiments, a therapeutically effective dose of anAAV vector results in FIX activity adequate to maintain hemostasis in asubject with hemophilia B, while producing no or minimal humoral (i.e.,antibody) immune response against the capsid, genome and/or Factor IXprotein produced from transduced cells. The antibody response to avirus, or virus-like particles such as AAV vectors, can be determined bymeasuring antibody titer in a subject's serum or plasma using techniquesfamiliar to those of skill in the field of immunology. Antibody titer toany component of an AAV vector, such as the capsid proteins, or a geneproduct encoded by the vector genome and produced in transduced cells,such as Factor IX Padua (or other FIX variant), can be measured usingsuch techniques. Antibody titers are typically expressed as a ratioindicating the dilution before which antibody signal is no longerdetectable in the particular assay being used to detect the presence ofthe antibody. Different dilution factors can be used, for example,2-fold, 5-fold, 10-fold, or some other dilution factor. Any suitableassay for the presence of an antibody can be used, for example andwithout limitation, ELISA, FACS, or a reporter gene assay, such asdescribed in WO 2015/006743. Use of other assays is also possibleaccording to the knowledge of those skilled in the art. Antibody titerscan be measured at different times after initial administration of anAAV vector.

In certain embodiments, a therapeutically effective dose of an AAVvector results in at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, ormore FIX activity in subjects with hemophilia B, while producing anantibody titer against the capsid, genome and/or Factor IX protein (suchas FIX Padua) produced from transduced cells that is not greater than1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200,1:300, 1:400, 1:500, or more, when determined at 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,12 months, 18 months, 2 years, 3 years, 4 years, 5 years, or a longerperiod after the subjects were administered the AAV vector. According toone exemplary non-limiting embodiment, an AAV vector results in at least20% FIX activity in a subject with severe hemophilia B while inducing anantibody titer against the capsid and/or Factor IX produced bytransduced cells that is not greater than 1:2, 1:3 or 1:4, both at 6months after the AAV vector was administered. In any of theseembodiments, the AAV vector can be AAV-FIX39-Padua, or an AAV vectorhaving the same capsid and a genome sequence at least 95%, 96%, 97%, 98%or 99% identical thereto, which may be administered to a subject in apharmaceutically acceptable composition alone, or with empty capsids ofthe same capsid type at an empty to vector ratio of about 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some other ratio.

As noted above, prior trials using AAV-mediated gene therapy forhemophilia B triggered a self-limiting immune response that preventedthe therapy from being effective for a significant period of timewithout need for high doses of steroids to cause immunosuppression. Animportant factor appears to have been a cellular immune response thateliminated the liver cells that had been transduced with the AAV vectorsunder study. This effect was detectable from both an elevation of liverenzymes, suggesting liver damage, and the presence of capsid-specific Tcells in the subjects.

In certain embodiments, a therapeutically effective dose of an AAVvector results in FIX activity adequate to maintain hemostasis in asubject with hemophilia B, while producing no or minimal cellular immuneresponse against the capsid and/or Factor IX protein produced fromtransduced cells. A cellular immune response can be determined in atleast two ways: assaying for T cell activity specific for capsidproteins or Factor IX, and testing for the presence of elevated liverenzyme levels that indicate damage to hepatocytes.

In some embodiments, cellular immune response is determined by assayingfor T cell activity specific for capsid proteins and/or the Factor IXprotein produced by the transduced liver cells. Different assays for Tcell response are known in the art. In one exemplary, non-limitingembodiment, T cell response is determined by collecting peripheral bloodmononuclear cells (PBMC) from a subject that was previously treated withan AAV vector for treating hemophilia B. The cells are then incubatedwith peptides derived from the VP1 capsid protein used in the vector,and/or the Factor IX protein, such as FIX Padua, produced by thetransduced liver cells. T cells that specifically recognize the capsidprotein or Factor IX protein will be stimulated to release cytokines,such as interferon gamma or another cytokine, which can then be detectedand quantified using the ELISPOT assay, or another assay familiar tothose of skill in the art. (See, e.g., Manno, et al., Nat Med 2006;12(3):342-347). T cell response can be monitored before and at differenttimes after a subject has received a dose of an AAV vector for treatinghemophilia B, for example, weekly, monthly, or some other interval.Thus, according to certain embodiments, a therapeutically effective doseof an AAV vector results in FIX activity adequate to maintain hemostasisin a subject with hemophilia B (for example, FIX activity of at least1%, 5%, 10%, 20%, 30%, or more), while causing a T cell response asmeasured using ELISPOT that is not greater than 10, 20, 30, 40, 50, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, or more,spot-forming units per 1 million PBMCs assayed when measured weekly,monthly, or some other interval after the AAV vector is administered, orat 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2years, or some different time after the AAV vector is administered. Insome of these embodiments, the ELISPOT assay is designed to detectinterferon gamma (or some other cytokine) production stimulated bypeptides from the AAV vector capsid protein or Factor IX protein(including FIX Padua, or a different variant) produced by transducedliver cells. In any of these embodiments, the AAV vector can beAAV-FIX39-Padua, or an AAV vector having the same capsid and a genomesequence at least 95%, 96%, 97%, 98% or 99% identical thereto, which maybe administered to a subject in a pharmaceutically acceptablecomposition alone, or with empty capsids of the same capsid type at anempty to vector ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, or some other ratio.

As a proxy for the cellular immune response against transducedhepatocytes, the presence of greater-than-normal liver enzymes can beassayed using standard methods. While not wishing to be bound by theory,it is believed that T cells specific for certain AAV vectors, such asthose used in prior clinical trials, can attack and kill transducedhepatocytes, which transiently releases liver enymes into thecirculation. Exemplary liver enzymes include alanine aminotransferase(ALT), aspartate aminotransferase (AST), and lactate dehydrogenase(LDH), but other enzymes indicactive of liver damage can also bemonitored. A normal level of these enzymes in the circulation istypically defined as a range that has an upper level, above which theenzyme level is considered elevated, and therefore indicactive of liverdamage. A normal range depends in part on the standards used by theclinical laboratory conducting the assay. In certain embodiments, atherapeutically effective dose of an AAV vector results in FIX activityadequate to maintain hemostasis in a subject with hemophilia B (forexample, FIX activity of at least 1%, 5%, 10%, 20%, 30%, or more), whilecausing an elevated circulating liver enzyme level, such as that of ALT,AST, or LDH, which is not greater than 0%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%,1000%, 1500%, 2000% of the upper limit of normal (ULN) value of theirrespective ranges, on average, or at the highest level measured inmultiple samples drawn from the same subject under treatment atdifferent times (e.g., at weekly or monthly intervals) afteradministration of the AAV vector. In any of these embodiments, the AAVvector can be AAV-FIX39-Padua, or an AAV vector having the same capsidand a genome sequence at least 95%, 96%, 97%, 98% or 99% identicalthereto, which may be administered to a subject in a pharmaceuticallyacceptable composition alone, or with empty capsids of the same capsidtype at an empty to vector ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, or some other ratio.

In prior clinical trials using AAV vectors to treat hemophilia B, theinvestigators needed to co-administer an immunosuppressant drug, such asa steroid, to prevent the subjects receiving treatment from mounting animmune response that would eliminate the transduced cells producing theFactor IX protein. Due to the attenuated immune response seen insubjects undergoing experimental treatment with certain AAV vectors,however, co-administration of immunosuppressing drugs may not benecessary. Thus, in certain embodiments, a therapeutically effectivedose of an AAV vector is one that is sufficient to maintain adequatehemostasis in a subject with severe or moderate hemophilia B, withoutneed for co-administration (before, contemporaneously, or after) of animmunosuppressant drug (such as a steroid or other immunosuppressant).Because an immune response is not predictable in all subjects, however,the methods herein of treatment for hemophilia B include AAV vectorsthat are co-administered with an immunosuppressant drug.Co-administration of an immunosuppressant drug can occur before,contemporaneously with, or after AAV vectors are administered to asubject having hemophilia B. In some embodiments, an immunosuppressantdrug is administered to a subject for a period of days, weeks, or monthsafter being administered an AAV vector for treating hemophilia B.Exemplary immunosuppressing drugs include steroids (e.g., withoutlimitation, prednisone or prednisolone) and non-steroidalimmunosuppressants, such as cyclosporin, rapamycin, and others. Whatdrug doses and time course of treatment are required to effectsufficient immunosuppression will depend on factors unique to eachsubject undergoing treatment, but determining dose and treatment timeare within the skill of those ordinarily skilled in the art. In someembodiments, an immunosuppressant may need to be administered more thanone time.

According to certain embodiments, a therapeutically effective dose of anAAV vector results in a consistent elevation of FIX activity whenadministered to a population of human subjects with severe or moderatehemophilia B. Consistency can be determined by calculating variabilityof response in a population of human subjects using statistical methodssuch the mean and standard deviation (SD), or another statisticaltechnique familiar to those of skill in the art. In some embodiments, atherapeutically effective dose of an AAV vector, when administered to apopulation of human subjects with severe or moderate hemophilia Bresults, at 3 months, 6 months, 9 months, 12 months, 15 months, 18months, 21 months, or 21 months after administration, in a mean FIXactivity of 1-5% with a SD of less than 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, or 1; a mean FIX activity of 2.5-7.5% with a SD of lessthan 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a mean FIXactivity of 5-10% with a SD of less than 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 7.5-12.5% with a SD ofless than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a meanFIX activity of 10-15% with a SD of less than 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 12.5-17.5% with a SDof less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; amean FIX activity of 15-20% with a SD of less than 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 17.5-22.5% witha SD of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1;a mean FIX activity of 20-25% with a SD of less than 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 22.5-27.5% witha SD of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1;a mean FIX activity of 25-30% with a SD of less than 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 27.5-32.5% witha SD of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1;a mean FIX activity of 30-35% with a SD of less than 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 32.5-37.5% witha SD of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1;a mean FIX activity of 35-40% with a SD of less than 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 37.5-42.5% witha SD of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1;a mean FIX activity of 40-45% with a SD of less than 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; a mean FIX activity of 42.5-47.5% witha SD of less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1;or a mean FIX activity of 45-50% with a SD of less than 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In any of these embodiments, theAAV vector can be AAV-FIX39-Padua, or an AAV vector having the samecapsid and a genome sequence at least 95%, 96%, 97%, 98% or 99%identical thereto, which may be administered to a subject in apharmaceutically acceptable composition alone, or with empty capsids ofthe same capsid type at an empty to vector ratio of about 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some other ratio.

Invention methods and uses can be combined with any compound, agent,drug, treatment or other therapeutic regimen or protocol having adesired therapeutic, beneficial, additive, synergistic or complementaryactivity or effect. Exemplary combination compositions and treatmentsinclude second actives, such as, biologics (proteins), agents and drugs.Such biologics (proteins), agents, drugs, treatments and therapies canbe administered or performed prior to, substantially contemporaneouslywith or following any other method or use of the invention, for example,a therapeutic method of treating a subject for a blood clotting disease.

The compound, agent, drug, treatment or other therapeutic regimen orprotocol can be administered as a combination composition, oradministered separately, such as concurrently or in series orsequentially (prior to or following) delivery or administration of anucleic acid, vector, recombinant vector (e.g., rAAV), vector genome, orrecombinant virus particle. The invention therefore providescombinations in which a method or use of the invention is in acombination with any compound, agent, drug, therapeutic regimen,treatment protocol, process, remedy or composition, set forth herein orknown to one of skill in the art. The compound, agent, drug, therapeuticregimen, treatment protocol, process, remedy or composition can beadministered or performed prior to, substantially contemporaneously withor following administration of a nucleic acid, vector, recombinantvector (e.g., rAAV), vector genome, or recombinant virus particle of theinvention, to a subject.

In certain embodiments, a combination composition includes one or moreimmunosuppressive agents. In certain embodiments a method includesadministering or delivering one or more immunosuppressive agents to themammal. In certain embodiments, a combination composition includesAAV-FIX particles and one or more immunosuppressive agents. In certainembodiments, a method includes administering or delivering AAV-FIXparticles to a mammal and administering an immunosuppressive agent tothe mammal. The skilled artisan can determine appropriate need or timingof such a combination composition with one or more immunosuppressiveagents and administering the immunosuppressive agent to the mammal.

Methods and uses of the invention also include, among other things,methods and uses that result in a reduced need or use of anothercompound, agent, drug, therapeutic regimen, treatment protocol, process,or remedy. For example, for a blood clotting disease, a method or use ofthe invention has a therapeutic benefit if in a given subject a lessfrequent or reduced dose or elimination of administration of arecombinant clotting factor protein to supplement for the deficient ordefective (abnormal or mutant) endogenous clotting factor in thesubject. Thus, in accordance with the invention, methods and uses ofreducing need or use of another treatment or therapy are provided.

The invention is useful in animals including human and veterinarymedical applications. Suitable subjects therefore include mammals, suchas humans, as well as non-human mammals. The term “subject” refers to ananimal, typically a mammal, such as humans, non-human primates (apes,gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal(dogs and cats), a farm animal (poultry such as chickens and ducks,horses, cows, goats, sheep, pigs), and experimental animals (mouse, rat,rabbit, guinea pig). Human subjects include fetal, neonatal, infant,juvenile and adult subjects. Subjects include animal disease models, forexample, mouse and other animal models of blood clotting diseases andothers known to those of skill in the art.

Subjects appropriate for treatment include those having or at risk ofproducing an insufficient amount or having a deficiency in a functionalgene product (protein), or produce an aberrant, partially functional ornon-functional gene product (protein), which can lead to disease.Subjects appropriate for treatment in accordance with the invention alsoinclude those having or at risk of producing an aberrant, or defective(mutant) gene product (protein) that leads to a disease such thatreducing amounts, expression or function of the aberrant, or defective(mutant) gene product (protein) would lead to treatment of the disease,or reduce one or more symptoms or ameliorate the disease. Targetsubjects therefore include subjects having aberrant, insufficient orabsent blood clotting factor production, such as hemophiliacs (e.g.,hemophilia B).

Subjects appropriate for treatment in accordance with the inventionfurther include those previously or currently treated with supplementalprotein (e.g., recombinant blood clotting factor such as FIX to treathemophilia). Subjects appropriate for treatment in accordance with theinvention moreover include those that have not developed a substantialor detectable immune response against FIX protein, or amounts ofinhibitory antibodies against FIX protein that would interfere with orblock FIX based gene therapy.

In other embodiments, human pediatric subjects that are determined tohave hemophilia B (e.g., by genotyping), but have not yet exhibited anyof the symptoms of hemophilia B, can be treated prophylactically with anAAV vector to prevent any such symptoms from occurring in the firstplace or, in other embodiments, from being as severe as they otherwisewould have been in the absence of treatment. In some embodiments, humansubjects treated prophylactically in this way are at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months old, or older, whenthey are administered an AAV vector to produce and maintain FIX activityadequate to maintain hemostasis, and thus prevent or reduce the severityof one or more symptoms of hemophilia B. In any of these embodiments,the AAV vector can be AAV-FIX39-Padua, or an AAV vector having the samecapsid and a genome sequence at least 95%, 96%, 97%, 98% or 99%identical thereto, which may be administered to a subject in apharmaceutically acceptable composition alone, or with empty capsids ofthe same capsid type at an empty to vector ratio of about 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some other ratio.

Administration or in vivo delivery to a subject can be performed priorto development of an adverse symptom, condition, complication, etc.caused by or associated with the disease. For example, a screen (e.g.,genetic) can be used to identify such subjects as candidates forinvention compositions, methods and uses. Such subjects thereforeinclude those screened positive for an insufficient amount or adeficiency in a functional gene product (protein), or that produce anaberrant, partially functional or non-functional gene product (protein).

Methods and uses of the invention include delivery and administrationsystemically, regionally or locally, or by any route, for example, byinjection or infusion. Such delivery and administration includeparenterally, e.g. intravascularly, intravenously, intramuscularly,intraperitoneally, intradermally, subcutaneously, or transmucosal.Exemplary administration and delivery routes include intravenous (i.v.),intraperitoneal (i.p.), intrarterial, subcutaneous, intra-pleural,intubation, intrapulmonary, intracavity, iontophoretic, intraorgan,intralymphatic.

Alternatively or in addition, AAV vector can be delivered to the livervia the portal vein. In another alternative, a catheter introduced intothe femoral artery can be used to deliver AAV vectors to liver via thehepatic artery. Non-surgical means can also be employed, such asendoscopic retrograde cholangiopancreatography (ERCP), to deliver AAVvectors directly to the liver, thereby bypassing the bloodstream and AAVneutralizing antibodies. Other ductal systems, such as the ducts of thesubmandibular gland, can also be used as portals for delivering AAVvectors into a subject that develops or has preexisting anti-AAVantibodies.

Doses can vary and depend upon whether the type, onset, progression,severity, frequency, duration, or probability of the disease to whichtreatment is directed, the clinical endpoint desired, previous orsimultaneous treatments, the general health, age, gender, race orimmunological competency of the subject and other factors that will beappreciated by the skilled artisan. The dose amount, number, frequencyor duration may be proportionally increased or reduced, as indicated byany adverse side effects, complications or other risk factors of thetreatment or therapy and the status of the subject. The skilled artisanwill appreciate the factors that may influence the dosage and timingrequired to provide an amount sufficient for providing a therapeutic orprophylactic benefit.

Methods and uses of the invention as disclosed herein can be practicedwithin 1-2, 2-4, 4-12, 12-24 or 24-72 hours after a subject has beenidentified as having the disease targeted for treatment, has one or moresymptoms of the disease, or has been screened and is identified aspositive as set forth herein even though the subject does not have oneor more symptoms of the disease. Of course, methods and uses of theinvention can be practiced 1-7, 7-14, 14-21, 21-48 or more days, monthsor years after a subject has been identified as having the diseasetargeted for treatment, has one or more symptoms of the disease, or hasbeen screened and is identified as positive as set forth herein.

Invention nucleic acids, vectors, recombinant vectors (e.g., rAAV),vector genomes, and recombinant virus particles and other compositions,agents, drugs, biologics (proteins) can be incorporated intopharmaceutical compositions, e.g., a pharmaceutically acceptable carrieror excipient. Such pharmaceutical compositions are useful for, amongother things, administration and delivery to a subject in vivo or exvivo.

As used herein the term “pharmaceutically acceptable” and“physiologically acceptable” mean a biologically acceptable formulation,gaseous, liquid or solid, or mixture thereof, which is suitable for oneor more routes of administration, in vivo delivery or contact. A“pharmaceutically acceptable” or “physiologically acceptable”composition is a material that is not biologically or otherwiseundesirable, e.g., the material may be administered to a subject withoutcausing substantial undesirable biological effects. Thus, such apharmaceutical composition may be used, for example in administering aviral vector or viral particle to a subject.

Such compositions include solvents (aqueous or non-aqueous), solutions(aqueous or non-aqueous), emulsions (e.g., oil-in-water orwater-in-oil), suspensions, syrups, elixirs, dispersion and suspensionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration or in vivo contact ordelivery. Aqueous and non-aqueous solvents, solutions and suspensionsmay include suspending agents and thickening agents. Suchpharmaceutically acceptable carriers include tablets (coated oruncoated), capsules (hard or soft), microbeads, powder, granules andcrystals. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration or delivery, as set forth herein orknown to one of skill in the art. Thus, pharmaceutical compositionsinclude carriers, diluents, or excipients suitable for administration byvarious routes.

Compositions suitable for parenteral administration comprise aqueous andnon-aqueous solutions, suspensions or emulsions of the active compound,which preparations are typically sterile and can be isotonic with theblood of the intended recipient. Non-limiting illustrative examplesinclude water, saline, dextrose, fructose, ethanol, animal, vegetable orsynthetic oils.

Cosolvents and adjuvants may be added to the formulation. Non-limitingexamples of cosolvents contain hydroxyl groups or other polar groups,for example, alcohols, such as isopropyl alcohol; glycols, such aspropylene glycol, polyethyleneglycol, polypropylene glycol, glycolether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acidesters. Adjuvants include, for example, surfactants such as, soyalecithin and oleic acid; sorbitan esters such as sorbitan trioleate; andpolyvinylpyrrolidone.

Pharmaceutical compositions and delivery systems appropriate for thecompositions, methods and uses of the invention are known in the art(see, e.g., Remington: The Science and Practice of Pharmacy (2003)20^(th) ed., Mack Publishing Co., Easton, Pa.; Remington'sPharmaceutical Sciences (1990) 18^(th) ed., Mack Publishing Co., Easton,Pa.; The Merck Index (1996) 12^(th) ed., Merck Publishing Group,Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms(1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel andStoklosa, Pharmaceutical Calculations (2001) 11^(th) ed., LippincottWilliams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug DeliverySystems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

A “unit dosage form” as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity optionally in association with apharmaceutical carrier (excipient, diluent, vehicle or filling agent)which, when administered in one or more doses, is calculated to producea desired effect (e.g., prophylactic or therapeutic effect). Unit dosageforms may be within, for example, ampules and vials, which may include aliquid composition, or a composition in a freeze-dried or lyophilizedstate; a sterile liquid carrier, for example, can be added prior toadministration or delivery in vivo. Individual unit dosage forms can beincluded in multi-dose kits or containers. Recombinant vector (e.g.,rAAV) sequences, vector genomes, recombinant virus particles, andpharmaceutical compositions thereof can be packaged in single ormultiple unit dosage form for ease of administration and uniformity ofdosage.

The invention provides kits with packaging material and one or morecomponents therein. A kit typically includes a label or packaging insertincluding a description of the components or instructions for use invitro, in vivo, or ex vivo, of the components therein. A kit can containa collection of such components, e.g., a nucleic acid, recombinantvector, virus (e.g., AAV) vector, vector genome or virus particle andoptionally a second active, such as another compound, agent, drug orcomposition.

A kit refers to a physical structure housing one or more components ofthe kit. Packaging material can maintain the components sterilely, andcan be made of material commonly used for such purposes (e.g., paper,corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer, lot numbers, manufacture location and date, expirationdates. Labels or inserts can include information identifyingmanufacturer information, lot numbers, manufacturer location and date.Labels or inserts can include information on a disease for which a kitcomponent may be used. Labels or inserts can include instructions forthe clinician or subject for using one or more of the kit components ina method, use, or treatment protocol or therapeutic regimen.Instructions can include dosage amounts, frequency or duration, andinstructions for practicing any of the methods, uses, treatmentprotocols or prophylactic or therapeutic regimes described herein.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, complications or reactions, such as warnings to the subject orclinician regarding situations where it would not be appropriate to usea particular composition. Adverse side effects or complications couldalso occur when the subject has, will be or is currently taking one ormore other medications that may be incompatible with the composition, orthe subject has, will be or is currently undergoing another treatmentprotocol or therapeutic regimen which would be incompatible with thecomposition and, therefore, instructions could include informationregarding such incompatibilities.

Labels or inserts include “printed matter,” e.g., paper or cardboard, orseparate or affixed to a component, a kit or packing material (e.g., abox), or attached to an ampule, tube or vial containing a kit component.Labels or inserts can additionally include a computer readable medium,such as a bar-coded printed label, a disk, optical disk such as CD- orDVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage mediasuch as RAM and ROM or hybrids of these such as magnetic/optical storagemedia, FLASH media or memory type cards.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control.

All of the features disclosed herein may be combined in any combination.Each feature disclosed in the specification may be replaced by analternative feature serving a same, equivalent, or similar purpose.Thus, unless expressly stated otherwise, disclosed features (e.g.,modified nucleic acid, vector, plasmid, a recombinant vector (e.g.,rAAV) sequence, vector genome, or recombinant virus particle) are anexample of a genus of equivalent or similar features.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a nucleic acid” includes a plurality of suchnucleic acids, reference to “a vector” includes a plurality of suchvectors, and reference to “a virus” or “particle” includes a pluralityof such virions/particles.

As used herein, all numerical values or numerical ranges includeintegers within such ranges and fractions of the values or the integerswithin ranges unless the context clearly indicates otherwise. Thus, toillustrate, reference to 80% or more identity, includes 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%,82.5%, etc., and so forth.

Reference to an integer with more (greater) or less than includes anynumber greater or less than the reference number, respectively. Thus,for example, a reference to less than 100, includes 99, 98, 97, etc. allthe way down to the number one (1); and less than 10, includes 9, 8, 7,etc. all the way down to the number one (1).

As used herein, all numerical values or ranges include fractions of thevalues and integers within such ranges and fractions of the integerswithin such ranges unless the context clearly indicates otherwise. Thus,to illustrate, reference to a numerical range, such as 1-10 includes 1,2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc.,and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., upto and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2,2.3, 2.4, 2.5, etc., and so forth.

Reference to a series of ranges includes ranges which combine the valuesof the boundaries of different ranges within the series. Thus, toillustrate reference to a series of ranges, for example, of 1-10, 10-20,20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250,250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000,2,000-2,500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500,4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000,includes ranges of 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000,etc.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments and aspects. The invention alsospecifically includes embodiments in which particular subject matter isexcluded, in full or in part, such as substances or materials, methodsteps and conditions, protocols, or procedures. For example, in certainembodiments or aspects of the invention, materials and/or method stepsare excluded. Thus, even though the invention is generally not expressedherein in terms of what the invention does not include aspects that arenot expressly excluded in the invention are nevertheless disclosedherein.

A number of embodiments of the invention have been described.Nevertheless, one skilled in the art, without departing from the spiritand scope of the invention, can make various changes and modificationsof the invention to adapt it to various usages and conditions.

Accordingly, the following examples are intended to illustrate but notlimit the scope of the invention claimed.

Example 1. Vector Design/Preparation

A novel Factor IX nucleic acid encoding a high specific activity humanfactor IX protein having the 338L Padua variant (Simioni P, et al., NEngl J Med 2009, 361:1671) was designed (“FIX 39-Padua”; SEQ ID No:10;FIG. 10). FIX39-Padua is completely devoid of CpG dinucleotides in theFIX coding and intronic sequences. For comparative testing, FIX19(Mingozzi et al. Sci. Transl Med. 2013) was prepared and modified toinclude the FIX Padua, to rule out any potential confounding effectsresulting from the FIX Padua (“FIX 19-Padua”; SEQ ID NO:11; FIG. 11).

A plasmid (“pAAV-ApoE_hAAT-FIX39”; 11125 bp; SEQ ID NO:12; FIG. 12A) wassynthesized and included the FIX39-Padua expression cassette and theelements described in Table 2. A map of pAAV-ApoE_hAAT-FIX39 is shown inFIG. 13.

TABLE 2 pAAV-ApoE_hAAT-FIX39 5′ AAV2 ITR SEQ ID NO: 13 Enhancer (HepaticSEQ ID NO: 14 Control Region) hAAT promoter SEQ ID NO: 15 5′ UTR SEQ IDNO: 16 FIX39-Padua CDS SEQ ID NO: 10 (FIG. 10) Intron A SEQ ID NO: 17(FIG. 14) 3′ UTR SEQ ID NO: 18 polyA SEQ ID NO: 19 3′ AAV2 ITR SEQ IDNO: 20 Lambda stuffer SEQ ID NO: 21 F1 origin of replication SEQ ID NO:22 Kanamycin resistance SEQ ID NO: 23 pUC origin of replication SEQ IDNO: 24

The sequence of the FIX39-Padua coding sequence and intron A is setforth in SEQ ID NO:25 (FIG. 15). A plasmid was also synthesized thatincluded the FIX19-Padua CDS and the same regulatory elements, the sameadeno-associated inverted terminal repeats (ITRs), and the sameliver-specific ApoE/hAAT promoter as pAAV-ApoE_hAAT-FIX39.

AAV vector having the 4-1 capsid variant (SEQ ID NO:4) was prepared forthe FIX39-Padua (“AAV-FIX39-Padua”) and FIX19-Padua (“AAV-FIX19-Padua”)transgenes using a triple transfection process followed by double cesiumchloride gradient centrifugation (Ayuso E, M et al., Gene Ther 2010,17:503). Vectors were titrated by quantitative PCR using a linearizedplasmid as the standard. For the study described in Example 3, vectorwas diluted in PBS, 5% sorbitol, 0.001% F68 to a final volume of 200 μlper mouse, for tail vein injection.

Example 2. In Vitro AAV Variant 4-1 Transduction

Primary hepatocytes from cynomolgus macaque and human origin weretransduced with the 4-1 variant capsid (SEQ ID NO:4) expressingluciferase at four different multiplicities of infection (MOI) rangingfrom 500 to 62,500 vector genomes per cell. Seventy-two hours aftertransduction, luciferase expression was analyzed. As shown in FIG. 16,the ratio of transduced human hepatocytes relative to non-human primatehepatocytes ranged from 0.8 to 1.5, depending on the MOI used. Thesedata generated in vitro appear to be consistent with previousobservations in vivo when comparing expression of coagulation factor IXin cynomolgus macaques and human subjects.

Example 3. Potency Study

A study was conducted to evaluate the potency of AAV-FIX39-Padua versusAAV-FIX19-Padua in mice. Groups of 5 mice were injected at 8-10 weeks ofage with either 1×10¹¹ or 1×10¹² vg/kg of AAV-FIX39-Padua andAAV-FIX19-Padua. Following vector administration, blood was collected byretro-orbital bleeding using heparinized capillary tubes; plasma wasisolated by centrifugation at 9000 rpm for 10 minutes at 4° C. andstored frozen at −80° C. until assayed.

Plasma collected was used to evaluate hFIX transgene expression. HumanFIX levels in plasma were measured using an ELISA kit (AffinityBiologicals, Ancaster, ON, Canada).

Activity levels of human FIX were measured by activated partialthromboplastin time (aPTT) assay. The aPTT assay was performed by mixingsample plasma in a 1:1:1 volume-ratio with human FIX-deficient plasma(George King Biomedical, Inc) and aPTT reagent (Trinity Biotech),followed by a 180s incubation period at 37° C. Coagulation was initiatedby addition of 25 mM calcium chloride. Time to clot formation wasmeasured using a STart 4 coagulation instrument (Diagnostica Stago). Astandard curve was generated with pooled normal plasma from George Kingstarting at a 1:5 dilution in TBS pH 7.4 (48 μl+192 μl) followed byserial 1:2 dilutions (120 μl+120 μl). The human standard curve was usedto calculate the activity of each sample at week 17 after vectoradministration; activity in two untreated mice was also measured. FIXactivity in untreated mice was averaged and then subtracted from thetreated samples to calculate the extra (i.e. human) activity due to theFIX Padua protein.

As shown in FIG. 17, AAV-FIX39-Padua and AAV-FIX19-Padua appear toexpress substantially equivalent levels of FIX.

Seventeen weeks after vector administration, human FIX activity wasmeasured in those mice treated with a vector dose of 1×10¹² vg/kg. Theactivity-to-antigen ratio ranged between 5.2 and 7.5, with an averagevalue of 6.4 for both FIX19-Padua and FIX39-Padua groups (Table 3).

TABLE 3 Human FIX activity values Antigen Activity Animal ID (% ofnormal) (% of normal) Ratio 01 - FIX19 53.9 352.7 6.5 02 - FIX19 95.6631.8 6.6 03 - FIX19 120.6 882.3 7.3 04 - FIX19 132.9 797.1 6.0 05 -FIX19 105.2 599.7 5.7 06 - FIX39 163.1 1092.8 6.7 07 - FIX39 108.2 670.36.2 08 - FIX39 121.1 781.2 6.4 09 - FIX39 152.3 1147.8 7.5 10 - FIX39134.1 702.1 5.2 Average AAV- 101.7 652.7 6.4 FIX19- Padua AAV- 135.8878.8 6.4 FIX39- Padua

While these results suggest that the potency of both expressioncassettes is substantially similar, the two constructs were alsoanalyzed in the setting of plasmid hydrodynamic tail vein injection. Therationale for evaluating FIX levels derived of in vivo administration ofnaked DNA was to compare both expression cassettes without the potentialinterferencE of differences in AAV tittering, vector manufacturing, etc.

As shown in FIG. 18, both naked expression cassettes were equally potentat driving FIX expression, confirming the data obtained in the AAVsetting. These results indicate that the FIX19-Padua and FIX39-Paduaexpression cassettes have similar potency.

Example 4. AAV-FIX39-Padua Gene Therapy

A clinical study is being conducted to determine safety and kinetics ofa single IV infusion of AAV-FIX39-Padua. The AAV 4-1 capsid variant usedhas been shown in preclinical studies to have good safety and efficacy,the ability to achieve sustained FIX activity levels of ˜35% in NHPs at1×10¹² vg/kg after 3 months of vector infusion; and cross reactingneutralizing antibodies (Ab) to the AAV 4-1 capsid variant areapproximately 10% less prevalent than AAV8. The design of the study isprovided in Table 4.

TABLE 4 AAV-FIX39-Padua Clinical Study Design Safety and Clinicallysignificant in vital signs, lab values and Tolerability of clinicalassessments (including number of bleeds AAVFIX39- Padua and QoL) frombaseline Kinetics of Transgene FIX activity levels and antigen levelsAAVFIX39-Padua at peak and steady-state Dosing Starting, Middle andHighest Dose Cohorts will each include 2-5 subjects Design Open-label,non-randomized, dose escalation Participating USA and potentiallyEurope, Japan and Canada countries Sample size Up to 15 subjectsEligibility Ages Eligible for Study: 18 Years and older Genders Eligiblefor Study: Male Accepts Healthy Volunteers: No Inclusion Able to provideinformed consent and comply Criteria with requirements of the studyMales ≥18 y.o. with confirmed diagnosis of hemophilia B (≤2 IU/dL or ≤2%endogenous factor IX) Received ≥50 exposure days to factor IX products Aminimum of an average of 4 bleeding events per year requiring episodictreatment of factor IX infusions or prophylactic factor IX infusions Nomeasurable factor IX inhibitor as assessed by the central laboratory andhave no prior history of inhibitors to factor IX protein Agree to usereliable barrier contraception until 3 consecutive samples are negativefor vector sequences Exclusion Evidence of active hepatitis B or CCriteria Currently on antiviral therapy for hepatitis B or C Havesignificant underlying liver disease Have serological evidence* of HIV-1or HIV-2 with CD4 counts ≤200/mm3 (*subjects who are HIV+ and stablewith CD4 count >200/mm3 and undetectable viral load are eligible toenroll) Have detectable antibodies reactive with 4-1 variant AAV capsid(SEQ ID NO: 4) Participated in a gene transfer trial within the last 52weeks or an investigational drug within the last 12 weeks Unable orunwilling to comply with study assessments Screening Visit Eligibilityevaluation AAV NAb titer is the major screen failure (highly recommendreferring subjects to CHOP's AAV NAb titer protocol for phone screening)Day 0 Visit FIX product incremental recovery then vector infusionFollow-up Visits Safety and kinetic evaluations (~17 visits) End-ofStudy Visit Final safety evaluation (at week 52)

Example 5. Clinical Results

Four subjects with hemophilia B were administered a single IV infusionof AAV-FIX39-Padua vector. The first two subjects, ages 23 and 18respectively, had no prior history of liver disease, while the third,age 47, had a history of HCV infection but had cleared spontaneously.All four subjects had been screened for neutralizing antibodies to thenovel AAV capsid and found to be negative.

Subjects were infused intravenously with 5×10¹¹ vg/kg of AAV-FIX39-Paduavector over a period of ˜1 hour. The total AAV-FIX39-Padua vectoradministered to each subject is shown in FIGS. 20-23, which had beencombined with the indicated amount of AAV empty capsids.

FIGS. 19-23 show study results, with AAV-FIX39-Padua vector administeredat day 0. The results show increased Factor IX production in all foursubjects, as reflected by increased FIX activity throughout the studyevaluation period.

The initial increase in FIX activity from day 0 to about day 3 is due toadministration of 100 IU/Kg Alprolix™ or BeneFIX™, which are recombinantFIX-Fc fusion protein having an approximate half-life of about 82 hours.Factor IX activity attributable to the AAV-FIX39 Padua vector begins atabout day 6-8 after AAV vector infusion.

As summarized in FIG. 19 and shown for each individual subject in FIGS.20, 21A, 22A and 23A, Factor IX activity gradually increased andappeared stable throughout the 183, 102, 69 and 50 day evaluationperiods for all four subjects. These data indicate that a singleinfusion of 5×10¹¹ vg/kg of AAV-FIX39-Padua vector results in sufficientand sustained Factor IX production and activity to provide hemophilia Bpatients with meaningful and beneficial blood clotting activity toprovide hemostasis.

As shown in FIGS. 19, 20A, 21A, 22A and 23A, Factor IX activity levelswere at 28%, 41%, 26% and 33% of normal, for subjects 1-4 respectively,at 183, 102, 69 and 50 days after infusion. Subject #3 treated himselfwith an extended half-life product for a suspected ankle bleed 2 daysafter vector infusion; other than this there have been no factorinfusions and no bleeds during the evaluation period.

Immunosuppressing agents (steroids) have not been administered to any ofthe subjects. In addition, in general there have been no sustainedelevations of transaminases above the upper limit of normal, indicatingno adverse effects of the treatment (FIGS. 20B, 21B, 22B and 23).

ELISPOTs were used to monitor T cell responses to AAV and to FIX in allfour subjects and have shown no or very low responses. Of note, the timecourse of rise in Factor IX levels to a plateau level has beenremarkably consistent to date (FIG. 19). Modest fluctuations in antigenlevels lead to greater shifts in activity levels, given the 8-foldincrease in specific activity of the Factor IX Padua variant.

Published data (Nathwani et al., N Engl J Med. 371(21):1994-2004 (2014))have shown long-term expression of Factor IX in men with hemophilia Binfused with an AAV8 vector expressing wild-type Factor IX. However,levels of expression were low-ranging from 1.4%-2.2% normal at thelowest dose (2×10¹¹ vector genomes [vg]/kg body weight) to 2.9-7.2% atthe highest dose (2×10¹² vg/kg). Moreover, 4/6 subjects infused at thehighest dose required a course of immunosuppressant (prednisolone) toreduce rising transaminases associated with the highest dose (but notobserved at lower doses of 2×10¹¹ or 6×10¹¹ vg/kg). Data from a naturalhistory study of patients with hemophilia suggest that circulatinglevels of −12% FIX are required to reduce the annual number ofspontaneous joint bleeds to zero (den Uijl et al., Haemophilia17(1):41-4 (2011)).

These are the first clinical results using a novel bioengineered AAVcapsid expressing a high specific activity Factor IX transgene. TheFactor IX activity levels seen in subjects 1-4 28%, 41%, 26% and 33% ofnormal are substantially greater circulating Factor IX levels than thethose seen in prior studies, based on published data, and exceed thecirculating Factor IX levels needed to reduce the annual number ofspontaneous joint bleeds to zero.

Furthermore, the substantial Factor IX activity levels seen in thisstudy were achieved with no recombinant Factor IX use since vectorinfusion, and without using immunosuppressing agents (steroids). Theseresults show the development of an AAV-FIX vector that can direct highlevel clotting factor expression at low doses of AAV vectoradministration, so that immunosuppression is not required—an importantgoal for liver-directed gene therapy. Factor IX activity levels observedin this study have been sustained over the duration of the study period.

Example 6. Reduced Immunogenicity of AAV-FIX39-Padua Vector

For the current Phase I/II study, four subjects receiving 5e11 vg/kg ofAAV-FIX39-Padua were monitored for potential immune responses againstthe AAV vector using a validated interferon-gamma (IFN-g) Enzyme LinkedImmunospot (ELISPOT) assay. Purified PBMCs isolated from weekly blooddraws were tested via interferon gamma ELISPOT assay. Six AAV capsidpeptide pools, containing 24-25 peptides each were incubated with 2e5cells in triplicate. T cell responses were detected using a biotinylatedantibody against IFN-g, followed by colorimetric development andreported as spot-forming units (SFU) per million cells. The highestresponding pool at each timepoint is shown as SFU/million cells. Thehistorically used cutoff for positivity is >50 SFU and 3-fold mediacontrol (blue line). Subjects 840-003-001, 840-001-002, 840-001-004, and840-001-005 (shown in black) have been followed as far out as weeks 26,14, 11, and 8 respectively. ELISPOT results from a previous trial inwhich two subjects, CP-16 and PT17 received 1e12 vg/kg of the AAV8-FIX19vector and one subject received 2e12 vg/kg of AAV8-FIX19, are shown inred.

Using the historically accepted value of >50 SFU and 3-fold thebackground (media) control as criteria for positive T cell response,there has been very little to no response in the three subjects as farout as 26-weeks post infusion (FIG. 24A). This is in stark contrast to apreviously unpublished study by our group using a codon optimized AAV8vector to deliver the FIX transgene cassette to 3 subjects, in whichrobust IFN-g T cell responses were observed as early as the week 2timepoint (FIG. 24B). Other previously published studies using AAV-2(Manno et al., 2006 Nat Med) and AAV-8 self-complementary vectors(Nathwani et al., 2011 NEJM) have also shown evidence of early T cellresponses to the AAV capsid as well. Importantly, no responses againstthe transgene product have been observed in this trial.

It is hypothesized that the activation of a T-cell mediated immuneresponse against transduced hepatocytes presenting AAV capsid T cellepitopes may play a role in subjects that show short-lived and eventualloss of transgene expression. Therefore, the reduced immunogenicityprofile of the AAV-FIX39-Padua vector represents a promising improvementtowards overall efficacy.

What is claimed:
 1. A method of treating a human subject with severe ormoderate hemophilia B comprising: administering to said subject atherapeutically effective amount of a recombinant adeno-associated virus(rAAV) vector comprising a vector genome encapsidated by an AAV capsid,wherein said vector genome comprises at least two AAV inverted terminalrepeats (ITR), an expression control element conferring livertissue-specific expression operably linked with a nucleic acid sequenceencoding human Factor IX (FIX) protein, and a polyadenylation signalsequence, wherein said nucleic acid sequence encoding human FIX proteinis at least 70% identical to SEQ ID NO:10, has a reduced number of CpGdi-nucleotides compared to wild-type nucleic acid sequence encodinghuman FIX protein and encodes the same human FIX protein encoded by SEQID NO:10, wherein said therapeutically effective amount of said rAAVvector is a dose of at least 5×10¹¹ vector genomes per kilogram (vg/kg)subject body weight, and is effective to reduce the severe or moderatehemophilia B to mild hemophilia B or a hemophilia B disease-free state.2. The method of claim 1, wherein said nucleic acid sequence encodinghuman FIX protein is at least 80% identical to SEQ ID NO:10.
 3. Themethod of claim 2, wherein said nucleic acid sequence encoding human FIXprotein is at least 85% identical to SEQ ID NO:10.
 4. The method ofclaim 1, wherein said vector genome further comprises an intron.
 5. Themethod of claim 4, wherein said intron is positioned within said nucleicacid sequence encoding human FIX protein.
 6. The method of claim 1,wherein said vector genome is linear single-stranded DNA.
 7. The methodof claim 1, wherein an AAV ITR is positioned at each end of the vectorgenome, and said expression control element comprises an enhancer and apromoter.
 8. The method of claim 7, wherein said AAV ITRs are AAV2 ITRs,said enhancer is a human apolipoprotein HCR enhancer, and said promoteris a human alpha-1-antitrypsin gene promoter.
 9. The method of claim 7,wherein said AAV capsid is selected from the group consisting of: (i) anAAV capsid of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, Rh10, or AAV-2i8; (ii) an AAV capsid comprising aVP1 protein consisting of the amino acid sequence of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ IDNO:9; (iii) an AAV capsid comprising a VP1 protein consisting of theamino acid sequence of SEQ ID NO:1 with an amino acid substitution atany one or more of amino acid positions 195, 199, 201 or 202 in SEQ IDNO:1; and (iv) an AAV capsid comprising a VP1 protein consisting of theamino acid sequence of SEQ ID NO:1 with an amino acid substitution ofarginine for any one or more lysines in SEQ ID NO:1.
 10. The method ofclaim 7, wherein said therapeutically effective amount of said rAAVvector is a dose ranging from 1×10¹² to 5×10¹³ vg/kg subject bodyweight, and wherein said AAV capsid is an AAV5 capsid.
 11. The method ofclaim 10, wherein said therapeutically effective amount of said rAAVvector is a dose selected from the group consisting of 1×10¹², 2×10¹²,3×10¹², 4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³,3×10¹³, 4×10¹³, and 5×10¹³ vg/kg subject body weight.
 12. The method ofclaim 11, wherein said therapeutically effective amount of said rAAVvector is a dose of 2×10¹³ vg/kg subject body weight.
 13. The method ofclaim 10, wherein said AAV ITRs are AAV2 ITRs, said enhancer is a humanapolipoprotein HCR enhancer, and said promoter is a humanalpha-1-antitrypsin gene promoter.
 14. The method of claim 13, whereinsaid therapeutically effective amount of said rAAV vector is a dose of2×10¹³ vg/kg subject body weight.
 15. The method of claim 13, whereinsaid vector genome further comprises an intron positioned between saidpromoter and said nucleic acid sequence encoding human FIX protein. 16.The method of claim 15, wherein said therapeutically effective amount ofsaid rAAV vector is a dose of 2×10¹³ vg/kg subject body weight.
 17. Themethod of claim 1, wherein said treatment is effective to produce anaverage FIX activity of at least 20% of normal for a sustained period ofat least 6 months.
 18. The method of claim 1, wherein said treatment iseffective to produce an average FIX activity of at least 30% of normalfor a sustained period of at least 12 months.
 19. The method of claim 1,wherein said treatment is effective to produce plasma FIX levels ofabout 0.06 to 0.50 IU/mL.
 20. The method of claim 1, wherein saidtreatment is effective to produce plasma FIX levels of about 0.06 to1.50 IU/mL.
 21. The method of claim 10, wherein said treatment iseffective to produce plasma FIX levels of about 0.06 to 0.50 IU/mL. 22.The method of claim 10, wherein said treatment is effective to produceplasma FIX levels of about 0.06 to 1.50 IU/mL.
 23. The method of claim12, wherein said treatment is effective to produce plasma FIX levels ofabout 0.06 to 0.50 IU/mL.
 24. The method of claim 12, wherein saidtreatment is effective to produce plasma FIX levels of about 0.06 to1.50 IU/mL.
 25. The method of claim 14, wherein said treatment iseffective to produce plasma FIX levels of about 0.06 to 0.50 IU/mL. 26.The method of claim 14, wherein said treatment is effective to produceplasma FIX levels of about 0.06 to 1.50 IU/mL.
 27. The method of claim1, wherein said treatment is effective to reduce the average frequencyof FIX replacement therapy needed to control bleeding by at least 80%.28. The method of claim 1, wherein said treatment is effective to reducethe average frequency of spontaneous bleeding episodes by at least 80%.29. The method of claim 1, wherein said treatment is effective toproduce average FIX activity of 30% to 50% of normal 12 months afteradministration with a standard deviation of 15% or less.
 30. The methodof claim 1, wherein said treatment does not cause circulating liverenzyme levels to exceed 100% of the upper limit of normal value.